Istaroxime-containing intravenous formulation for the treatment of acute heart failure (ahf)

ABSTRACT

Compositions for intravenous infusion of istaroxime, or a metabolite of istaroxime, in human patients suffering from heart failure are disclosed. Likewise, methods for extended infusion of istaroxime or its metabolites in individuals with heart failure are disclosed. In particular, some methods disclosed herein include the infusion of istaroxime, or a metabolite thereof, for a period of time that is greater than six hours in order to improve cardiac relaxation without triggering arrhythmogenic events in an individual suffering from heart failure. Other methods include administration of istaroxime until certain plasma concentration thresholds of istaroxime metabolites are achieved. Also disclosed are istaroxime metabolites with selective SERCA2a activation.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of PCT/US19/60961, filed Nov. 12, 2019, whichclaims benefit of the filing date of U.S. Provisional Application No.62/814,149, filed Mar. 5, 2019, the entire contents of each of which areincorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the field of pharmaceuticals, inparticular to an istaroxime-containing intravenous formulation for usefor the treatment of acute heart failure.

BACKGROUND OF THE INVENTION

The prevalence of heart failure (HF) is age-dependent, ranging from lessthan 2% of people younger than 60 years to more than 10% of people olderthan 75 years (Metra M, Teerlink J R, Lancet 2017; 390:1981-1995). Mostpatients with HF have a history of hypertension, coronary arterydisease, cardiomyopathies, or valve disease, or a combination of thesedisorders (Metra M, Teerlink J R, Lancet 2017; 390:1981-1995). Thecalculated lifetime risk of developing HF is expected to increase andthose with hypertension are at higher risk (Lloyd-Jones D M et al.,Circulation 2002; 106:3068-3072). Patients with HF have a poor prognosiswith high rates of hospital admission and mortality.

Clinical symptoms in HF are caused by a cardiac double pathologicalfeature that consists in an inotropic abnormality, resulting indiminished systolic emptying (systolic dysfunction) and a complianceabnormality in which the ability of the ventricles to suck blood fromthe venous system is impaired (diastolic dysfunction), thus reducing theamount of blood available for systolic contraction, which is animpairment of left ventricle (LV) filling. Whatever the initialtriggering mechanism of HF, an abnormal distribution of intracellularCa²⁺ resulting from reduced Ca²⁺ uptake by the sarcoplasmic reticulum(SR), which is the main intracellular Ca²⁺ store (Schwinger R H et al.,J Mol Cell Cardiol. 1999; 31(3):479-91; Bers D et al., Ann N.Y. Acad Sci2006; 1080:165-177), underlies the impaired contractility andrelaxation. This abnormal Ca²⁺ distribution involves the Ca²⁺-ATPase ofthe SR membrane (SERCA2a), an ATP dependent Ca²⁺ transport pump. SERCA2aactivity is physiologically limited by its interaction withphospholamban (PLN) (Asahi M et al., J Biol Chem 1999; 274: 32855-32862;Toyoshima C et al., Proc Natl Acad Sci U S A 2003; 100: 467-47; Bers DM., Annu Rev Physiol 2008; 70:23-49.; MacLennan D H, Kranias E G., NatRev Mol Cell Biol 2003; 4(7): 566-577), SERCA2a restraint is normallyrelieved by PLN phosphorylation by protein kinase A (PKA), a signallingpathway severely depressed as a consequence of HF remodelling (Karim C Bet al., J Mol Biol 2006; 358: 1032-1040; Lohse M et al., Circ Res 2003;93:896-906; Bers D M, Physiology 2006; 21: 380-387; Mann D L, Bristow MR, Circulation 2005; 111:2837-2849). A deficiency in cardiac SERCA2aactivity is widely recognized as the main cause of the reduced Ca²⁺uptake in the SR of the failing myocardium (Bers D et al., Ann N.Y. AcadSci 2006; 1080:165-177; Bers D M, Physiology 2006; 21: 380-387;Minamisawa S et al., Cell 1999; 99: 313-322.).

In addition to its consequences on myocyte contractility and relaxation,abnormal Ca²⁺ distribution also facilitates cardiac arrhythmias (Zaza A& Rocchetti M, Curr Pharm Des 2015; 21:1053-1061) and, on the long term,it accelerates myocytes loss by apoptosis (Nakayama H et al., J ClinInvest 2007; 117:2431-44). Reduced SERCA2a function also increases theenergy cost of contraction, because it requires a compensatory increasein Ca²⁺ extrusion through the Na—Ca exchanger (NCX), which is lessenergy efficient (Lipskaya L et al., Expert Opin Biol Ther 2010;10:29-41). Substantial evidence indicates that normalization of SERCA2afunction restores intracellular Ca²⁺ homeostasis and improvescontractility and relaxation of cardiomyocytes and of the heart in situ(Byrne M J et al., Gene Therapy 2008; 15:1550-1557; Sato et al., J BiolChem 2001; 276:9392-99). To summarize, recovery of SERCA2a function inHF may improve cardiac relaxation and contractility while minimizingarrhythmias, myocardial oxygen consumption and myocyte death (Lipskaia Let al., Expert Opin Biol Ther. 2010; 10:29-41). In parallel to SERCA2aactivation, inhibition of the Na,K-pump can further increaseintracellular Ca²⁺ content without inducing excessive cytosolic Ca²⁺accumulation (Shattock M J et al., J Physiol. 2015; 15; 593(6):1361-82).Therefore, the combination of Na,K-ATPase inhibition and SERCA2astimulation may afford further positive inotropy at a reduced risk ofarrhythmogenic Ca²⁺ triggering events.

Current long-term therapy of HF is centred on prevention of “myocardialremodelling” and neuro-hormonal storm (β-blockers, ACE inhibitors,aldosterone antagonists), which is a chronic maladaptive response toreduced contractility, amplifies the initial damage and underliesdisease evolution (Heineke J & Molkentin D, Nat Rev 2006; 7:589-600).While this approach has indisputable merit, it does not target impairedheart “contractility” and “relaxation”, which are the functionalderangements defining HF and responsible for its symptoms. Indeed,particularly in the advanced disease stages, such as in patients withacute heart failure (AHF), drugs that increase myocardialcontractility/relaxation (“inotropic/lusitropic agents”) are stillwidely used and crucial for the management of patients with AHF (MetraM, Teerlink J R, Lancet 2017; 390:1981-1995). These includesympathomimetic amines (dobutamine) and levosimendan, a Ca²⁺-sensitizerwith a strong vasodilator effect. Unfortunately, these agents act bymechanisms with potentially harmful components, such as facilitation oflife-threatening arrhythmias, increased myocardial oxygen consumptionand, in some patients, impairment of an already insufficient coronaryblood flow due to the fall in blood pressure caused by vasodilatation(Ashkar H & Makaryus A N, Dobutamine [updated 2018 Oct. 27], InStatPearls [Internet], Treasure Island (FL): StatPearls Publishing,2018-January-2017 (available athttps://www.ncbi.nlm.nih.gov/books/NBK470431/); Gong B. et al., JCardiothorac Vasc Anesth 2015; 29:1415-25; EDITORIAL Patel P A et al.,Circ Heart Failure 2014; 7:918-925). This limits the use of these agentsfor relieving the symptoms of the AHF, as clearly stated in both the USand EU guidelines that assign to them and evidence grade C, which is thelowest level of evidence based on the results of the available clinicaltrials (Rigopoulus A G et al., Herz 2017 Sep. 22; Butler J et al., Eur JHeart Fail. 2018; 20(5):839-841; Georghiade M et al., J Am Coll Cardiol.2008; 51:2276-85). Furthermore, these agents do not improve patient'sprognosis and survival, and their therapeutic use must be carefullymonitored (Ashkar H & Makaryus A N, Dobutamine [updated 2018 Oct. 27],In StatPearls [Internet], Treasure Island (FL): StatPearls Publishing,2018 January-2017 (available athttps://www.ncbi.nlm.nih.gov/books/NBK470431/); Gong B. et al., JCardiothorac Vasc Anesth 2015 29:1415-25).

Among positive inotropes, the cardiac glycoside Digoxin, which is aninhibitor of the Na,K-ATPase enzymatic activity, has been one of themost commonly prescribed medications in the past. However, its use hasbeen decreasing over the last decades because of the difficulty inmaintaining digoxin serum concentration ranges at which digoxin displaysits beneficial effects (0.5-0.7 ng/ml) without reaching the thresholdlevel of 0.9 ng/ml, above which is observed an increased risk of deathdue mainly to arrhythmias (Packer M, Journal of Cardiac Failure 2016;22:726-730; Packer M, Eur J Heart Failure 2018; 20:851-852). OMECAMTIVMECARBIL, a cardiac myosin activator that increases cardiac contractionwithout improving the impaired relaxation, is under clinicaldevelopment, but its cardiac effects are also associated with anincrease of high sensitive troponin plasma levels that indicates somedegree of cardiomyocytes injury/damages (Teerlink J R et al., J Am CollCardiol. 2016; 67(12):1444-1455).

Intensive research is also in progress for the development of HF drugswith mechanisms of action other than positive inotropy. The agents mostinvestigated and under clinical development are: SERELAXIN—recombinantrelaxin 2 mediator; ULARITIDE—recombinant natriuretic peptide;BMS986231—NO donor; ADRECIZUMAB—Adrenomedullin inhibitor;ANX-042—spliced variant of NP; TD1439—Neprylisin (NEP) inhibitor.However, when evaluated in clinical phase 2-3 trials, none of these newagents has met the primary end-point.

The clinical course and prognosis of a patient with chronic heartfailure (CHF) is much worse after an episode of AHF (Solomon S D et al.,Circulation 2007; 116:1482-87; Teneggi Vet al., Hear Failure Rev 2018;23:667-691). AHF can be defined as the new onset or recurrence ofsymptoms and signs of heart failure, requiring urgent evaluation andtreatment and resulting in unscheduled care or hospital admission(Teneggi V et al., Heart Failure Rev 2018; 23:667-691; Packer M, Eur JHeart Failure 2018; 20:851-852). Half of the patients with AHF havereduced systolic function (HFrEF), representing a target for potentialtherapies (Braunwald E., Lancet 2015; 385:812-24). Therapies for AHF inpatients with reduced ejection fraction (HFrEF) have focused onalleviating congestion with vasodilators, diuretics, or ultrafiltrationor increasing cardiac output with positive inotropes. Although thistherapeutic strategy has reduced the risk of sudden cardiac death, thepost-discharge event rate remains unacceptably high in patientshospitalized for AHF. Many unwanted cardiovascular side effects can becaused by the available therapy, such as myocardial ischemia, cardiacinjury and arrhythmias consequent to the inotrope therapy, particularlyin patients with coronary artery disease (CAD) (Abraham W T et al., J AmColl Cardiol 2005; 46:57-64; Flaherty J D et al., J Am Coll Cardiol.2009; 53(3):254-63), hypotension and low perfusion of the peripheralorgans (kidney) caused by vasodilators particularly in HF patients withlow blood pressure. Accordingly, the main goal during hospitalization isto improve cardiac output without causing cardiac and/or kidney injury.

Moreover, there has been little focus on examining or treating animpaired left ventricular (LV) diastolic relaxation that, in theremaining 50% of patients with HF but preserved (50) ejection fraction(HFpEF) or mid-range (40-49) reduction ejection fraction (HFmrEF orHFmEF), is responsible for the symptoms of HF (Butler J et al., Eur JHeart Fail. 2018; 20, 839-841; Bonsu K O et al., Heart Failure Reviews2018; 23:147-156). In addition, patients with AHF who have reduced EFalso exhibit an impairment of ventricular relaxation that contributes tothe overall failure of cardiac function. A variety of echocardiographicindexes has been developed to measure the cardiac relaxation capacityboth in animal models and patients with HF (e.g., decreased early mitralannular tissue velocity [e′] and decreased early mitral inflow [E]deceleration time [DT]), along with echocardiographic parameters ofincreased LV filling pressure (e.g., E/e′ ratio). Even though thecorrespondence of the single index changes is not perfectlysuperimposable in some animal models and patients, their overall changesin animal models of ventricular relaxation impairment are certainlytranslatable to the human condition and used to study the drug effect inAHF (Shah S A et al., Am Heart J 2009; 157:1035-41).

Various therapeutic approaches that increase SERCA2a function have beenpreviously investigated. These include SERCA2a overexpression by genetransfer (Byrne et al., Gene Therapy 2008; 15:1550-1557) or PLNinactivation by expression of mutants with negative dominance (HoshijimaM et al., Nat. Med. 2002; 8:864-871; Iwanaga Y et al., J Clin Invest2004; 113, 727-736), AdV-shRNA (Suckau L et al., Circulation 2009;119:1241-1252), microRNA (Gröβl Tet al., PLoS One 2014; 9:e92188) orantibodies (Kaye D M et al., J. Am. Coll. Cardiol. 2007; 50:253-260). Ashighlighted by the negative results of the largest phase 2b clinicaltrial applying SERCA2a gene delivery in HF (CUPID 2), these approachessuffer from major problems in construct delivery (viral vectors etc.)and dose adjustment that are far from being solved (Hulot J S, Eur HeartJ 2016; 19:1534-1541). A small-molecule (pyridone derivative)attenuating the inhibitory effect of phospholamban on SERCA2a activity,which is structurally different from istaroxime, has been recentlydescribed (Kaneko M et al., Eur J Pharmacol 2017; 814:1-7), but no dataon patients are available.

From the overall picture of the state of the art, and in spite of morethan 30 years of trials and related publications, the treatment ofpatients admitted to hospital because of AHF symptoms is still largely“opinion based” rather than being “evidence based” (Rigopoulus A G etal., Herz 2017 Sep. 22; Butler J et al., Eur J Heart Fail. 2018;20(5):839-841; Georghiade M et al., J Am Coll Cardiol. 2008;51:2276-85). Many of the available drugs were designed with rescue andsymptom relief in mind and not necessarily to target and correct anyspecific underlying pathophysiology/biochemical mechanism that may beresponsible for the symptoms of AHF.

As a general paradigm, drugs are molecules that produce their wanted orunwanted effect by interacting with the molecules/proteins of patients.The therapeutic benefits of these drugs depend upon their selectivity incorrecting the abnormalities of the protein underlying the diseasesymptoms over other possible effects on proteins with misappropriated oreven counterbalancing activities.

The deficiency in cardiac SERCA2a activity is widely recognized as oneof the most important causes of the decreased relaxation ofcardiomyocytes and increased susceptibility to arrhythmias in patientswith cardiac failure (Bers D et al., Ann N.Y. Acad Sci 2006;1080:165-177; Bers D M, Physiology 2006; 21:380-387; Minamisawa S etal., Cell 1999; 99:313-322; Fernandez-Tenorio M & Niggli E., J Mol CellCardiol. 2018 June; 119:87-95). To this end, the potential energystarved failing heart status may further potentiate the consequences ofSERCA2a deficiency (Ventura-Clapier R, Garnier A, Veksler V, Joubert F.,Biochim Biophys Acta. 2011 July; 1813(7):1360-72; Pinz I et al., J BiolChem 2011; 286(12):10163-10168). In turn, these two causes, ifacknowledged, may be adequately addressed. Moreover, for more than 200years (first evidence in literature: No Author listed, An Account of theEffects of the Digitalis Purpurea in Dropsy, Lond Med J. 1785; 6:55-60),Digitalis, which was subsequently recognized to act throughout theinhibition of the Na—K pump, has been used to increase cardiac pumpingactivity in spite of some unwanted side effects (e.g., arrhythmias orlong term cardiomyocytes damage) (Hougen T J, Friedman W F., Am JPhysiol. 1982 October; 243(4):H517-22; Whitbeck M G et al., Eur Heart J.2013 May; 34(20):1481-8). The latter effects are very likely due to theincreased cardiomyocytes cytoplasmic Ca²⁺ that, on one hand is usefulfor stimulate contraction but, on the other, may favor the abovementioned side effect that are further enhanced by the deficiency ofSERCA2a activity (Zaza A & Rocchetti M, Curr Parm Des 2015:21:1053-1061). Consequently, drugs with a combined “selective” effect onthese two molecular targets may be beneficial to patients or, at least,may prove or disprove the clinical impact of these two molecularmechanisms.

Notwithstanding the differences in the therapeutic response and outcomeof subsets of patients having different degrees of deficiency inrelaxation or contraction (Butler J, Eur J Heart Fail. 2018; 20,839-841;Bonsu K O et al., Heart Failure Reviews 2018; 23:147-156) (consideringthe parameters HFrEF HF, HFmEF or HFpEF Heart Failure reduced EjectionFraction (=<40), Heart Failure moderate reduction (m or mr) EjectionFraction (between 40 and 50) Heart Failure preserved Ejection Fraction(>50)), it is mandatory to develop therapeutic strategies aimed atassessing the proper combination of the two activities on SERCA2aactivation and Na—K pump inhibition for the three subsets of patients.

In particular, there is a strong and, to date, unmet need to improve thetherapy of acute heart failure in HFpEF patients (Bonsu K O et al.,Heart Failure Reviews 2018; 23:147-156) for whom an improvement ofdiastolic function by correcting the underlying molecular mechanism hasnot been yet achieved.

Istaroxime (PST 2744) is disclosed in EP0825197 and in De Munari S. etal., J. Med. Chem. 2003, 64, 3644-3654 and is the compound(3Z,5α)-3-[(2-aminoethoxy)imino]androstane-6,17-dione. Istaroxime is anew small-molecule drug under clinical development for the treatment ofAHFS that is endowed of the double mechanism of action of inhibiting theNa⁺/K⁺ pump (Micheletti R et al., J Pharmacol Exp Ther 2002;303:592-600) while activating SERCA2a (Rocchetti M et al., J PharmacolExp Ther 2005; 313:207-15).

At the same level of inotropy, the proarrhythmic effect of istaroxime isconsiderably lower than that of digoxin, a pure Na—K pump inhibitor(Rocchetti M et al., J Pharmacol Exp Ther. 2005; 313:207-15). Thissuggests that, by improving Ca²⁺ clearance from the cytosol (Alemanni, JMol Cell Cardiol 2011; 50:910-8), SERCA2a stimulation may also minimizethe proarrhythmic effect of Na—K pump blockade (Rocchetti M et al., JPharmacol Exp Ther 2005; 313:207-15; Zaza A & Rocchetti, M Curr PharmDes 2015; 21:1053-1061) while preserving its inotropic effect. Thereduction of the proarrhythmic effect by istaroxime has been confirmedin clinical studies (Georghiade M et al., J Am Coll Cardiol 2008;51:2276-85), wherein istaroxime was administered as a continuous 6-hourinfusion.

In HF patients, istaroxime infusion improved both systolic and diastolicfunctions. Amelioration of systolic function was detected as increasesin contraction tissue velocity (s′) and in the slope of end-systolicelastance (ESPVR slope); increased diastolic compliance was revealed byan increment in the early relaxation tissue velocity (e′) and decreasedend-diastolic elastance (EDPVR slope) (Shah S A et al., Am Heart J 2009;157:1035-41).

According to the results described in the Horizon study by Gheorghiade(Gheorghiade M et al., J Am Coll Cardiol 2008; 51:2276-85), whereistaroxime has been infused for 6 hours, the plateau effect on theimprovement of diastolic relaxation, continuously measured as a decreasein PCWP (pulmonary capillary wedge pressure), occurs after 3 hours ofinfusion, after which the level of PCWP remains constant up to 6 hours.As it may be expected from the parallel dual targets, SERCA2a and theNa—K pump, there is no clear separation between the effects on theechocardiographic indexes of relaxation and those of contraction whenincreasing the infusions doses of istaroxime. Therefore, the potentialbeneficial effect due to the SERCA2a activation cannot be separated fromthe potential detrimental effect due to the Na—K pump inhibition whenIstaroxime is infused up to 6 hours. Even though both the clinical(Gheorghiade M et al., J Am Coll Cardiol. 2008; 51:2276-85; Shah S A etal., Am Heart J 2009; 157:1035-41) and experimental studies in dog(Mattera G G et al., Am J Cardiol 2007; 99[suppl]:33A-40A) havedemonstrated that the presence of the SERCA2a-stimulating activity ofistaroxime considerably reduces the pro-arrhythmic activity associatedto the Na—K pump inhibition, studies are still not satisfactory as tothe clinical outcome, in particular to ensure a properly improveddiastolic function and a safer discharge of the patient from hospital.As a matter of fact, the Gheorghiade and Shah clinical trials(Gheorghiade M et al., J Am Coll Cardiol 2008; 51:2276-85; Shah S A etal., Am Heart J 2009; 157:1035-41) found that, at the end of the 6 hoursinfusion, traditional parameters of LV systolic performance, such asstroke volume index (SVI) and Ejection fraction (EF), did not changedramatically with istaroxime compared to placebo and the duration of theeffect on the diastolic relaxation was not properly discussed.

The overall HORIZON study where istaroxime has been infused for 6 hours(see Gheorghiade M et al., J Am Coll Cardiol. 2008; 51:2276-85; Shah S Aet al., Am Heart J 2009; 157:1035-41) showed a greater improvement ofsystolic contraction than of diastolic relaxation within a dose range of0.5 μg/Kg/min and 1.5 μg/Kg/min.

An improved diastolic function is expected to be achieved by a “pure”SERCA2a activator. However, notwithstanding the intense research ondiscovering small molecules or gene therapy aimed at selectivelyactivating SERCA2a, no promising clinical outcomes have been reached sofar.

Accordingly, there is a long-felt need for an advance in the treatmentof acute heart failure, in particular for improving diastolic function.The present invention satisfies the above needs and overcomes theproblem of prior art.

SUMMARY OF THE INVENTION

It has surprisingly been found that the intravenous infusion ofistaroxime for a time longer than 6 hours and up to 48 hours or moreprovides unexpected improvements to the cardiac diastolic relaxationechocardiographic indexes with respect to the same infusion for 6 hoursor less, while the echocardiographic indexes of systolic contraction arealmost unchanged from 6 hours to 24 hours of infusion time.

Described herein are pharmaceutical compositions containing istaroximeformulated for intravenous infusion in a human subject for use in atreatment method for acute heart failure. In particular, theadministration by infusion is for a duration longer than 6 hours,whereby the diastolic relaxation is improved as compared toadministration of istaroxime by intravenous infusion for a duration of 6hours or less (e.g., from 3 to 6 hours). In some embodiments, thediastolic relaxation improvement is measured by echocardiographicparameter E/A, E/e′ or by pulmonary capillary wedge pressure. In someembodiments, the infusion duration is up to about 24 hours. In others,it is up to about 36 or 48 hours. In such embodiments, thepharmaceutical composition containing istaroxime is administered at adose between 0.2 μg/kg/min and 1.5 μg/kg/min; preferably it isadministered at a dose between 0.25 μg/kg/min and 1.0 μg/kg/min.

In some embodiments, the pharmaceutical composition containingistaroxime is administered for a duration sufficient to produce a plasmalevel of an istaroxime metabolite that is greater than about 5 ng/ml forat least about 6 hours. In particular, the istaroxime metabolites maycomprise a formula (II) or a formula (III) compound, such as:

In some embodiments, the human subject suffers from heart failure withpreserved ejection fraction (HFpEF) or mid-range reduction ejectionfraction (HFmEF) and/or undergoing a therapeutic treatment for heartfailure with one or more further therapeutically active ingredients.

Another aspect of the invention features a compound having a formula(II) or formula (III):

In some embodiments, the compounds having the formula (II) or (III) areused in the treatment of a disease requiring the activation of SERCA2a,such as a cardiovascular disease. In particular, the compounds may beused to treat acute heart failure. In some aspects, these compounds areincluded in an admixture with at least one pharmaceutically acceptablevehicle and/or excipient.

Also disclosed herein are methods for treating an individual havingheart failure that include the steps of (1) providing an individualhaving heart failure; (2) administering a therapeutically effectiveamount of a pharmaceutical composition containing istaroxime to theindividual for an infusion duration of longer than 6 hours; and (3)measuring one or more parameters of heart function, such as diastolicrelaxation. In such methods, the administering of the pharmaceuticalcomposition results in an improvement in diastolic relaxation ascompared to istaroxime administered by intravenous infusion for 6 hoursor less, thereby treating the individual having acute heart failure. Inpreferred embodiments, the individual is human. In particularembodiments, the infusion duration is up to about 24 hours. In otherembodiments, the infusion duration is up to about 36 hours. In stillothers, the infusion duration is up to about 48 hours. In such methods,the istaroxime may be administered at a dose of about 0.2 μg/kg/min toabout 1.5 μg/kg/min. Preferably, it is administered at a dose of about0.25 μg/kg/min to about 1.0 μg/kg/min.

In some embodiments, the individual is diagnosed with heart failure withpreserved ejection fraction (HFpEF) or mid-range reduction ejectionfraction (HFmEF). In other embodiments, the individual is undergoing atherapeutic treatment for heart failure with one or more furthertherapeutically active ingredients.

Also featured herein are pharmaceutical compositions containingistaroxime for intravenous infusion in an individual for the treatmentof acute heart failure. In such aspects, the administration is for aninfusion duration sufficient to produce in the individual a plasmaconcentration level of an istaroxime metabolite that is greater thanabout 5 ng/ml for an accumulation period of at least about 6 hours,whereby the diastolic relaxation is improved as compared toadministration of istaroxime prior to the accumulation period.Preferably, the istaroxime metabolite comprises formula (II) or (III).

In some embodiments, the infusion duration is sufficient to produce aplasma concentration level of istaroxime metabolite that is greater thanabout 10 ng/ml for an accumulation period of at least about 6 hours. Inother embodiments, the plasma concentration level of the istaroximemetabolite is greater than about 15 ng/ml for an accumulation period ofat least about 6 hours. In still others, it is greater than about 20ng/ml. In some embodiments, the compositions are administered at a doseof about 0.2 pg/kg/min to about 1.5 μg/kg/min; preferably from about0.25 μg/kg/min to about 1.0 μg/kg/min).

Other aspects of the invention feature methods for treating anindividual having heart failure, including the steps of: (1) providingan individual having heart failure; (2) administering a therapeuticallyeffective amount of a pharmaceutical composition containing istaroximeto the individual for an infusion duration sufficient to produce aplasma concentration level of an istaroxime metabolite that is greaterthan about 5 ng/ml for an accumulation period of at least about 6 hours;and (3) measuring one or more parameters of heart function, wherein theone or more parameters of heart function comprises diastolic relaxation.

In some embodiments, the plasma concentration level of the istaroximemetabolite is greater than about 10 ng/ml for an accumulation period ofat least about 6 hours. In other embodiments, it is greater than about15 ng/ml for an accumulation period of at least about 6 hours. In stillothers, it is greater than about 20 ng/ml for an accumulation period ofat least about 6 hours. The therapeutically effective does of thecomposition is between about 0.2 μg/kg/min and about 1.5 μg/kg/min;preferably, between 0.25 μg/kg/min and 1.0 μg/kg/min.

In yet another aspect of the invention, a method for treating anindividual having acute heart failure is featured that includes thesteps of (1) providing an individual having acute heart failure; (2)measuring one or more parameters of heart function, wherein the one ormore parameters of heart function comprises diastolic relaxation asmeasured by echocardiographic parameter E/A, E/e′ or by pulmonarycapillary wedge pressure; and (3) administering a therapeuticallyeffective amount of a pharmaceutical composition containing istaroximeto the individual at a dose of at least about 1.0 μg/kg/min for aduration sufficient to improve diastolic relaxation in the individual.Then, after improvement of diastolic relaxation in the individual, atherapeutically effective amount of a second pharmaceutical compositioncontaining istaroxime is administered to the individual at a dose ofbetween about 0.25 μg/kg/min to about 1.0 μg/kg/min for an infusionduration sufficient to produce in the individual a plasma concentrationlevel of an istaroxime metabolite that is greater than about 5 ng/ml foran accumulation period of at least about 6 hours. In such aspects, theistaroxime metabolite comprises formula (II) or (III).

Still other aspects of the invention feature methods for treating anindividual having heart failure that include (1) providing an individualhaving heart failure; (2) administering a therapeutically effectiveamount of a pharmaceutical composition containing an istaroximemetabolite to the individual for an infusion duration of longer than 6hours; and (3) measuring one or more parameters of heart function, suchas diastolic relaxation. In some aspects, the infusion duration isbetween 6 hours and 48 hours at a dose of between about 0.2 82 g/kg/minto about 1.5 μg/kg/min; preferably between about 0.25 μg/kg/min to about1.0 μg/kg/min. In a particular embodiment, the individual is diagnosedwith heart failure with preserved ejection fraction (HFpEF) or mid-rangereduction ejection fraction (HFmEF).

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features and advantages of the present disclosure willbecome better understood with regard to the following description,appended claims, and accompanying drawings wherein:

FIG. 1 shows a time course of changes in pulmonary capillary wedgepressure (PCWP) in patients infused with placebo (white diamond) ascompared to istaroxime infusion for 6 hours at a dose of 0.5 μg/kg/min(dark diamond), 1.0 μg/kg/min (square), and 1.5 μg/kg/min (triangle).The X-axis represents the average PCWP (mmHg), and the Y-axis representstime (hours).

FIG. 2A shows the plasma levels of istaroxime (square) and itsmetabolites in Caucasian patients intravenously infused with 0.5μg/kg/min istaroxime for 24 hours. The metabolites are PST 2915(diamond), PST 2922 (circle), and PST 3093 (triangle). The X-axisrepresents the plasma concentration (ng/ml), and the Y-axis representstime (hours).

FIG. 2B shows the plasma levels of istaroxime (square) and itsmetabolites in Caucasian patients (left panel) and Chinese patients(right panel) intravenously infused with 0.5 μg/kg/min istaroxime for 24hours. The metabolites are PST 2915 (diamond), PST 2922 (circle), andPST 3093 (triangle). The X-axis represents the plasma concentration(ng/ml), and the Y-axis represents time (hours).

FIG. 2C shows the plasma levels of istaroxime (square) and itsmetabolites in Chinese patients intravenously infused with 1.0 μg/kg/ministaroxime for 24 hours. The metabolites are PST 2915 (diamond), PST2922 (circle), and PST 3093 (triangle). The X-axis represents the plasmaconcentration (ng/ml), and the Y-axis represents time (hours).

DETAILED DESCRIPTION OF THE INVENTION

The compositions and methods disclosed herein confer to individualssuffering from heart failure unexpected benefits. Provided herein arecompositions comprising istaroxime or a metabolite thereof. Further, asdisclosed herein, infusion with istaroxime or its metabolites for morethan 6 hours improves selectively cardiac relaxation over cardiaccontraction. Moreover, istaroxime infusion time can be extended to allowfor the accumulation of one or more of its metabolites, at least one ofwhich exhibits single-function SERCA2a activation (i.e., behaves as a“pure” SERCA2a activator). The compositions and methods disclosed hereinwill be described in more detail below.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood by one of ordinaryskill in the art to which this invention belongs. Standard techniquesare used unless otherwise specified. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods and examples areillustrative only, and are not intended to be limiting. Allpublications, patents and other documents mentioned herein areincorporated by reference in their entirety.

As used herein, the singular forms “a,” “an,” and “the” include theplural referents unless the context clearly indicates otherwise.

The term “about” refers to the variation in the numerical value of ameasurement, e.g., volume, time, pressure, concentration, etc., due totypical error rates of the device used to obtain that measure. In oneembodiment, the term “about” means within 5% of the reported numericalvalue, preferably, the term “about” means within 3% of the reportednumerical value.

The term “heart failure” refers to a clinical syndrome characterized bytypical symptoms (e.g., breathlessness, ankle swelling and fatigue) thatmay be accompanied by signs (e.g., elevated jugular venous pressure,pulmonary crackles and peripheral edema) caused by a structural and/orfunctional cardiac abnormality, resulting in a reduced cardiac outputand/or elevated intracardiac pressures at rest or during stress.

The terms “acute heart failure” or “AHF” are used interchangeably hereinand refer generally to a rapid onset or worsening of symptoms and/orsigns of HF requiring immediate treatment and hospitalization. Thecurrent definition of “acute heart failure” is rather nonspecific andmay include a broad spectrum of conditions with several phenotypescharacterized by different clinical presentation, etiology,precipitating factors, therapeutic approach, and prognosis. In addition,a large proportion of patients have a subacute course of the diseasewith a progressive worsening of signs and symptoms of HF which coulddevelop days before hospital admission.

The terms “chronic heart failure” or“CHF” are used interchangeablyherein and refer to the current clinical classification of chronic HFbased on the presence of signs and symptoms of HF and left ventricularejection fraction (LVEF), recognizing three categories: “heart failurewith reduced ejection fraction” or “HFrEF,” which is characterized by anLVEF of less than about 40%; “heart failure with mid-range ejectionfraction” or “HFmEF” or “HFmrEF,” which is characterized by an LVEF fromabout 40% to about 49%; and “heart failure with preserved ejectionfraction” or “HFpEF,” which is characterized by an LVEF of equal to orgreater than about 50%. The terms “HFmrEF” and “HFpEF” include twoadditional criteria, namely increased natriuretic peptides levels(BNP>35 μg/ml and/or NT-proBNP>125 μg/mL) associated with the evidenceof structural and/or functional heart disease (left ventricularhypertrophy and/or left atrium enlargement and/or evidence of diastolicdysfunction). The efficacy of HF evidence-based medications have beenconfirmed only in patients with “HFrEF,” whereas no treatmentdemonstrated a significant improvement of outcomes in patients with“HfpEF”.

The term “treating” refers to any indicia of success in the treatment oramelioration of the disease or condition. Treating can include, forexample, reducing or alleviating the severity of one or more symptoms ofthe disease or condition, or it can include reducing the frequency withwhich symptoms of a disease, defect, disorder, or adverse condition, andthe like, are experienced by an individual, such as a human patient.

The term “preventing” refers to the prevention of the disease orcondition, e.g., acute heart failure, in an individual, such as a humanpatient. For example, if an individual at risk of developing heartfailure is treated with the methods of the present invention and doesnot later develop heart failure, then the disease has been prevented inthat individual.

The term “treat or prevent” is sometimes used herein to refer to amethod that results in some level of treatment or amelioration of thedisease or condition, and contemplates a range of results directed tothat end, including but not restricted to prevention of the conditionentirely.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which an istaroxime compound or a metaboliteof istaroxime may be combined and which, following the combination, canbe used to administer the compound to a mammal.

As used herein, the term “pharmaceutically acceptable” salt, solvate,hydrate, or ester means a salt, solvate, hydrate, or ester form of theactive ingredient which is compatible with any other ingredients of thepharmaceutical composition, which is not deleterious to the subject towhich the composition is to be administered.

The term “intravenous infusion” refers to the administration or deliveryof liquid substances directly into a vein of a mammal. Typical“infusions” use only the pressure supplied by gravity.

The term “parameter” as used herein to refer to measuring heart functionmeans any heart function that is observable or measurable using suitablemeasuring techniques available in the art. A non-limiting list ofexemplary “parameters” of heart function include heart rate, bloodpressure, diastolic relaxation, systolic contraction, LVEF, diastolicblood pressure, systolic blood pressure, cardiac output, stroke volume,deceleration slope, cardiac index, mitral inflow velocity, and the like.As one having ordinary skill in the art will appreciate, measuring oneor more “parameters” of heart function can be used to detect heartdysfunction as compared to the average normal parameters and can also beused to determine whether heart function has improved following orduring treatment.

The terms “therapeutically active” or “active” ingredient or compoundrefer to a substance that provides a beneficial effect to the individualto whom the substance is administered. A “therapeutically effectiveamount” or “therapeutically effective dose” is the amount of acomposition or active ingredient sufficient to provide a beneficialeffect to the individual to whom the composition or active ingredient isadministered.

Description

The present invention is based on the unexpected discovery thatistaroxime infusion for more than 6 hours provides a prevailinglusitropic effect or improvement of the reduced cardiac relaxation, asshown by the clear changes of the echo indexes of cardiac relaxation(E/A DTs, e′, E/e′ and left atrial area or volume) while those ofcontraction (Sa and s) remained unchanged. In some embodiments, theistaroxime infusion is between about 6 hours and up to 48 hours or more,e.g., 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h, 40 h, 41h, 42 h, 43 h, 44 h, 45 h, 46 h, 47 h, 48 h, or more. For instance, theinfusion can be for up to 24 hours, or up to 36 hours, or up to 48hours, or more. In other embodiments, the duration of the infusion isgreater than 6 hours and equal to or less than about 48 hours, orgreater than 6 hours and equal to or less than about 36 hours, orgreater than 6 hours and equal to or less than about 24 hours, orgreater than 6 hours and equal to or less than about 12 hours. It beingunderstood that the infusion or istaroxime or a metabolite thereofprovides a prevailing lusitropic effect or improvement of the reducedcardiac relaxation as compared to infusion with istaroxime or ametabolite thereof for a period of 6 hours or less, e.g., 0, 1, 2, 3, 5,or 6 hours.

Importantly, an analysis of four independent groups of data confirmsthat istaroxime infusion for more than 6 hours provides a prevailinglusitropic effect and improvement of the cardiac relaxation. First, FIG.1 shows a PCWP time course of patients intravenously infused withistaroxime for up to 6 hours. As previously described in the HORIZONstudy of patients intravenously infused with istaroxime for 6 hours,time course changes of PCWP, which the skilled artisan will appreciateas a valid marker of diastolic relaxation, shows an improvement ofdiastolic relaxation during the initial 3 hours, but then the averagePCWP plateaus and remains unchanged for the following 3 hours (see FIG.1). In other words, patients infused for 6 hours with istaroxime showedno improvement in diastolic relaxation between 3 and 6 hours of theinfusion. In contrast, patients administered intravenous infusion ofistaroxime for 24 hours exhibit a clear increase in echocardiographicindexes of diastolic relaxation from 6 to 24 hours, while the indexes ofsystolic contraction remain unchanged (see Tables 1A-1C). Moreover, asshown in FIGS. 2A-2C, there is a progressive and remarkable increase inthe plasma concentration of the istaroxime metabolite PST 3093 at the6-hour and 24-hour time points, while the plasma concentration ofistaroxime remains constant throughout this time interval. Finally, thesynthesis of PST 3093 and the subsequent biochemical and pharmacologicalstudies demonstrate that this compound is endowed of a selective SERCA2astimulatory activity (see, e.g., Example 2 and Table 3) atconcentrations well below the that of the intravenous infusion studiesshown in FIG. 2. Moreover, its infusion in a rat model of diabeticcardiomyopathy is associated with an improvement in diastolic relaxation(see Table 5). According to Munafò et al. (Nature 2018,553(7689):399-401), simply repeating single experiments is notsufficient, but rather many lines of evidence is needed. Thus, it is theconsistency among these four independent findings that, per se, confersscientific robustness to the assertion that istaroxime infusion for morethan 6 hours provides a prevailing lusitropic effect and improvement ofthe cardiac relaxation as compared to shorter infusion times.

These findings are entirely unexpected, since the plasmatic level ofistaroxime remains constant (i.e., within about 10 ng/ml at 3 hour timepoint to about 8 ng/ml) during the following time points for the wholeduration of the infusion. The present inventors have discovered that inhumans this prolonged infusion of istaroxime generates an increasingconcentrations of istaroxime metabolites PST 3093 and PST 2915, whichbehave as selective or “pure” SERCA2a activator. Further, thisselectively is even greater for PST 3093, thus explaining the unexpectedeffect in improving diastolic function over systolic function.

Consequently, patients with HFpEF or HFmEF may benefit from a moreselective correction of the impaired diastolic relaxation by increasingthe plasma level of the metabolite over that of istaroxime. Thus,minimizing the Na⁺/K⁺ pump inhibition, notwithstanding the constantplasma level of istaroxime, with its associated unwanted effects in termof arrhythmias or cardiomyocytes damage. Advantageously, the clinicaloutcome is a safer patient discharge after treatment of acute heartfailure.

Istaroxime is an inotropic compound having the following structuralformula (I):

Upon administering to a mammal, such as a human, istaroxime ismetabolized into several metabolites that are capable of activatingSERCA2a.

Istaroxime metabolic pathway is illustrated below:

As such, disclosed herein are metabolites of istaroxime having SERCA2aactivity that have the following structural formulas (II) and (III):

In preferred embodiments, the metabolite of istaroxime (here also namedPST 3093) endowed with selective or “pure” SERCA2a activity is thecompound of formula (III).

The present inventors have isolated and characterized PST 2915 and PST3093. In rats with diabetic cardiomyopathy, administration of PST 3093showed improved diastolic relaxation and overall cardiac function asmeasured as an increase of stroke volume SV.

Therefore, it is also an object of the present invention to utilize theSERCA2a-activation properties of the compound of formula (II) or thecompound of formula (III), or their respective pharmaceuticallyacceptable salts or esters, as well as their different hydrates,solvates, polymorphic forms. Another object of the present invention isthe compound of formula (II) or formula (III) for use as medicament, inparticular for treating diseases requiring the activation of SERCA2a,more preferably for the treatment of heart failure or acute heartfailure.

Also disclosed herein is a pharmaceutical composition comprising thecompound of formula (II) or formula (III), in an admixture with at leastone pharmaceutically acceptable vehicle and/or excipient. In preferredembodiments, the pharmaceutical composition is formulated foradministering to an individual by infusion, preferably, it is byintravenous infusion.

The surprising effect of the present invention on the improved cardiacdiastolic relaxation can be better appreciated from the comparison ofcardiac parameters between the 6-hour infusion of the previous study(HORIZON clinical trial) and the 24-hour infusion according to thepresent invention. The outline of the study is described below and thedata at 6 hours of infusion, 24 hours of infusion, and 48 hours ofinfusion are summarized in Tables 1A, 1B, and 1C, respectively.

The synopsis of the clinical trial is described herein:

The clinical study of the safety and efficacy of Istaroxime in Treatmentof Acute Decompensated Heart Failure-A multicenter, randomized, double-Title blind, placebo controlled, parallel group clinical study.Indication Acute Decompensated Heart Failure (ADHF) Objective To Assessthe safety, tolerability and efficacy of two different doses ofistaroxime (0.5 and 1.0 μg/kg/min), a new agent with lusitropic andinotropic activities that improves the cardiac contraction- relaxationcycle. The 2 doses of istaroxime (0.5 and 1.0 μg/kg/min) will be infusedi.v. for 24 hours in comparison with placebo, in treatment of Chineseand Caucasian patients with Acute Decompensated Heart Failure. In allthe Caucasian patients and in a subset of Chinese patientspharmacokinetics and metabolism of istaroxime shall also be studied.Study Design A multicenter, randomized, double-blind,placebo-controlled, parallel group study. Study Period This studyincludes a screening period (Days −1), a treatment period (Day 1), apost-treatment period (Days 2-4), and a follow-up period (which includesone patient visit on Day 30). Subject Inclusion criteria SelectionPatients who fulfill the following inclusion criteria at screening willbe Criteria considered for the study: 1. Signed informed consent; 2.Male or female patients 18-85 years (inclusive); 3. Admission for arecurrent ADHF episode with dyspnea at rest or minimal exertion and needof intravenous diuretic therapy (≥40 mg iv. furosemide); 4. Systolicblood pressure between 90 and 125 mmHg (limits included) without signsor symptoms of hypoperfusion including cardiogenic shock, coldextremities and peripheral vasoconstriction, oliguria/anuria, signs ofcerebral hypo perfusion such as confusion; 5. Left ventricular (LV)Ejection fraction (EF) ≤40% measured by 2D-Echocardiography 6. E/Earatio >10 7. BNP 350 pg/mL or NT-pro-BNP ≥1400 pg/mL 8. Adequateechocardiography window (defined as visualization of at least 13/16segment of the left ventricle); Exclusion Criteria Any of the followingcriteria established at screening would render a patient ineligible forthe study: 1. Pregnant or breast-feeding women (women of child bearingpotential must have the results of a negative pregnancy test recordedprior to study drug administration) 2. Current (within 12 hours prior toscreening) or planned (through the completion of study drug infusion)treatment with any iv. therapies, including vasodilators (includingnitrates or nesiritide), positive inotropic agents and vasopressors 3.Current or need of mechanical support (intra-aortic balloon pump,endotracheal intubation, mechanical ventilation, or any ventricularassist device), 4. Ongoing treatment with oral digoxin. Patient treatedwith digoxin cannot be randomized. However, if digoxin treatment hasbeen stopped during the last week before randomization and the digoxinplasma level is <0.5 ng/ml, patient may be randomized; 5. History ofhypersensitivity to the study medication or any related medication 6.Diagnosis of cardiogenic shock within the past month; 7. Acute coronarysyndrome or stroke within the past 3 months; 8. Coronary artery bypassgraft or percutaneous coronary intervention within the past month orplanned in the next month; 9. Primary hypertrophic or restrictivecardiomyopathy or systemic illness known to be associated withinfiltrative heart disease; 10. Cor pulmonale or other causes ofright-sided HF not related to left ventricular dysfunction; 11.Pericardial constriction or active pericarditis; 12. Atrial fibrillationwith marked irregularities of heart rhythm; 13. Life threateningventricular arrhythmia or ICD (implantable cardioverter defibrillator)shock within the past month; 14. CRT (cardiac resynchronizationtherapy), ICD or pacemaker implantation within the past month; 15.Valvular disease as primary cause of HF; 16. Heart rate >120 bpm or <50bpm 17. Acute respiratory distress syndrome or ongoing sepsis; 18.Fever >38° 19. History of bronchial asthma or porphyria; 20. Donation orloss of blood equal to or exceeding 500 mL, during the 8 weeks beforeadministration of study medication; 21. Positive testing for Hepatitis Band/or Hepatitis C with abnormal liver functions; 22. Participation inanother interventional study within the past 30 days; 23.The followinglaboratory exclusion criteria, verified based on results obtained withinthe last 24 hours of hospitalization: a. Serum creatinine >3.0 mg/dl(>265 μmol/L); b. Aspartate aminotransferase (ASAT) or alanineaminotransferase (ALAT) >3 × upper limit of normal, c. Hemoglobin (Hb)<10 g/dL, d. Platelet count <100,000/μL, e. Serum potassium >5.3 mmol/Lor <3.8 mmol/L, Study Drugs Test drug: Istaroxime (10 mg per vial) Mode1 Intravenous infusion via a syringe pump. administratior TreatmentTreatment by i.v. infusion will last 24 hours. duration DosingIstaroxime 0.5-1.0 μg/kg/min since the beginning. A continuous i.v.scheme infusion for 24 hours not exceeding 144 mg for 24 hours ofistaroxime for patients with body weight >100 kg shall be carried out.Sample Size 120 total patients (96 Chinese patients and 24 Caucasianpatients) Study Screening period (between Hours −24 to −1) ProceduresWithin a maximum of 24 hours before administration of study medication(istaroxime), a medical screening will be performed on all prospectivepatients to assess suitability for the study. Prior to conducting anystudy specific procedures, the investigator or his/her designee willexplain the study fully to the patient and provide him/her with a copyof the Patient Information Sheet and Informed Consent Document. If thepatient is willing to participate in the study, s/he and theinvestigator or his/her designee will both sign the Informed ConsentDocument and a copy of the signed document will be kept by the patient.Treatment period (Day 1) 1) Confirm eligibility; 2) Randomization ofpatients (after eligibility has been confirmed) 3) Insertion of multiplelumen intravenous catheter 4) Start istaroxime or placebo infusion (dateand time of infusion start must be recorded in the CRF) 5) cTnT (atpre-dose: two samples, then at 3 and 6, 12, 24, 48 and 72 hours afterstart of infusion) 6) NT pro-BNP at baseline and at the end of 24 hoursinfusion 7) Blood samples collection for metabolites and PK (atpre-dose, 0.5-3-6- 12-24 after start of infusion and at 0.25, 0.5, 1, 412, 24 hours after the end of infusion) in all the Italian patients andin a subset of Chinese patients pharmacokinetics and metabolism ofistaroxime shall also be studied. 8) Vital signs (including bodytemperature and dyspnoea at pre-dose, 3, 6, 12 and 24 after the start ofinfusion) 9) 12-lead ECG profile 10) Stop Day −1 Holter 11) Start24-hour Holter ECG (Day 1 recording; to be started immediately beforeinitiation of the study drug infusion) 12) Echocardiography at baselineand 6 and 24 hours after infusion start 13) 24-hours urine collectionsfor measurement of istaroxime and its metabolites and urinary creatininefor the calculation of the creatinine clearance; 14) Blood collectionfor K + and eGFR between 23 hours and 30 minutes and 23 hours and 55minutes since infusion start; 15) Concomitant medication monitoring(including chronic medication; dose, date and time must be recorded onCRF) 16) Adverse events monitoring Post-treatment period (Day 2 to Day4) Evaluations at 24 hours (day 2) from randomization include: 1) Vitalsigns (including body temperature and dyspnea); 2) 12-lead ECG (singleECGs); 3) Stop 24-hour Holter ECG; 4) Start 24-hour Holter ECG (Day 2recording); 5) Stop istaroxime infusion (date and time of infusion endmust be recorded in the CRF); 6) Serum potassium level and 24-hour urinecollection for measurement of istaroxime metabolites and urinarycreatinine for calculation of the creatinine clearance; 7) Serumcreatinine clearance and calculation of eGFR; 8) cTnT (50% or 20%relative increase over the basal cTnT levels, respectively for patientswith cTnT basal levels < or > of the 99% URL (upper reference levels, asdefined for the Roche hs test, in patients with normal renal function,eGFR ≥ 85 ml/min); in patients with eGFR below this value, the renalfunction variations must be considered in evaluating the significance ofthe cTnT changes); 9) NT pro-BNP; 10) Metabolites; 11) Echocardiography;12) Concomitant medication monitoring (including chronic medication mustbe recorded in the CRF); 13) Adverse Events monitoring. Evaluations at48 hours (day 3) include: 1) Vital signs (including body temperature anddyspnea); 2) 12-Lead ECG (single ECGs); 3) Stop 24-hour Holter ECG; 4)Standard hematology; 5) Standard blood chemistry; 6) Serum potassiumlevel; 7) 24-h urine collection for measurement of istaroximemetabolites and urinary creatinine for the calculation of creatinineclearance ; 8) Calculation of eGFR; 9) NT-proBNP; 10) cTnT; 11) Bloodsamples for istaroxime metabolites; 12) Echocardiography 13) Adverseevents monitoring; 14) Concomitant medication monitoring (includingchronic medication must be recorded in the CRF); Evaluations at 72 hours(day 4) include: 1) cTnT and NTproBNP (at 72 hours after start ofinfusion) 2) Vital signs (including body temperature and dyspnoea) 3)Physical examination (HF signs included) 4) 12-lead ECG 5) Adverseevents monitoring 6) Concomitant medication monitoring (includingchronic medication) 7) Istaroxime metabolites 8) Serum potassium andcreatinine levels for calculation of eGFR 9) Creatinine clearanceFollow-up period and visit (Day 5 to Day 30) During the follow-up periodthe Investigator/designee will make every effort to establish patientoutcomes. Evaluations on Day 30 (follow-up visit) include: 1) Vitalsigns (including body temperature and dyspnoea); 2) 12-lead ECG intriplicate; 3) Calculation of eGFR; 4) Standard hematology; 5) Standardblood chemistry; 6) NT-proBNP 7) cTnT; 8) Urine pregnancy test (β-HCG)for females of childbearing potential 9) Urinalysis; 10) Physicalexamination (HF signs included); 11) Adverse events monitoring; 12)Concomitant medication monitoring (including chronic medication)Efficacy Efficacy endpoints Parameters 1. Primary efficacy end-point:Change from baseline to 24 hours after infusion start (treatment periodDay 1) in the E/Ea ratio assessed by tissue Doppler. 2. Secondaryefficacy end-points: Change from baseline to 24 hours in the treatmentperiod Day1 (addressing the differences between the changes at 6 and 24hours from baseline) of the following Echo-Doppler parameters: LVEjection fraction (EF) LV end systolic and end diastolic volumes Strokevolume index (SVI) E, A and E/A ratio Difference between the changes at6 and 24 hours from baseline of the Tissue Doppler parameter E/Ea OthersTissue Doppler parameters such as Sa, Da and Aa Changes in dyspnoeaassessed at 3, 6, 12, 24, 48 hours after infusion start by Visual AnalogScale (VAS) (including only patients presenting dyspnoea at baseline);Area under the curve (AUC) on changes in dyspnoea assessed at 3, 6, 12,24, 48 hours after infusion start by VAS (including only patientspresenting dyspnea at baseline); Changes in BNP from baseline at 24hours; Proportion of patients with hospital readmissions or emergencyvisits for cardiovascular reasons by Day 30; Proportion of patients withepisodes of worsening HF defined by the need to increase the dose orreinitiate i.v. therapy with diuretics and/or other inotropic agentsduring the hospitalization; Length of the hospitalization; Safety Safetyendpoints: Parameters The following safety endpoints will be assessedduring treatment and the post-treatment/follow-up periods: Incidence ofadverse events; Change in vital signs (including body temperature anddyspnoea); Change in 12-lead ECG parameters; Incidence of clinically orhemodynamically significant episodes of supraventricular or ventriculararrhythmias detected by continuous ECG dynamic monitoring; Change inlaboratory parameters (hematology, blood chemistry and urinalysis);Change in renal function; Change in in cTnT; Incidence of cTnT elevation(>50% or >20% relative increase over the basal cTnT levels at baseline,for patients with cTnT levels at baseline < or ≥ of the 99% URL (upperreference levels, as defined for the Roche hs test, in patients withnormal renal function, eGFR ≥ 85 ml/min); in patients with eGFR belowthis value, the renal function variations must be considered inevaluating the significance of the cTnT changes); Mortality at Day 30;PK Full plasma and urine PK profile: parameters The following PK metricswill be computed for E and Z isomers (when applicable) of istaroximeplasma concentrations using non-compartmental analysis: Cmax, tmax,AUC0-t, AUC0-∞, □z, t½, CIT, MRT, Vss, Vz; the following PK metrics willbe computed for E and Z isomers (when applicable) of istaroxime urineconcentrations: Ae, A0/0, CIR; In addition, the following PK metricswill be computed as above for plasma and urine concentrations of the Eand Z isomers (when applicable) of istaroxime metabolites 2915, 2922,and 3093: Cmax, tmax, AUC0-t, AUC0-∞, □z, t12 and, if possible, Ae andA0/0; Statistical Primary efficacy endpoint: Analysis The primaryefficacy endpoint (change from baseline in E/Ea ratio) will be analyzedusing a linear mixed model for repeated measures including treatment,centre, timepoint, gender, baseline cTnT (normal <URL, abnormal ≥URL),atrial fibrillation (Yes/No) and treatment*timepoint interaction asfixed effects and baseline and baseline*timepoint interaction ascovariates. The primary comparison will be 0.5 μg/kg/min dose ofistaroxime versus placebo at 24 hours. Highest dose of istaroxime (1.0μg/kg/min) versus placebo will be tested as a secondary comparison.Additional analyses separated by cohort will be implemented forsensitivity purpose. Secondary efficacy endpoints The followingsecondary endpoints: Change from baseline to 24 hours (addressing thedifferences between the changes at 6 and 24 hours from baseline) of thefollowing Echo-Doppler parameters: LV Ejection fraction (EF) LV endsystolic and end diastolic volumes Stroke volume index (SVI) E, A andE/A ratio Change from baseline to 24 hours in the E/Ea ratio assessed bytissue Doppler (difference between the changes at 6 and 24 hours frombaseline) Others Tissue Doppler parameters such as Sa, Da and Aa Changesin dyspnoea using VAS score will be analysed using a mixed model forrepeated measures similar to the one used for the primary efficacyendpoint. AUC on changes in dyspnoea by VAS and changes in BNP will beanalyzed using an ANCOVA model with treatment, centre, gender, baselinecTnT (normal <URL, abnormal ≥URL) and atrial fibrillation (Yes/No) asfixed effects and baseline dyspnea as covariate. Number and proportionof patients with: hospital readmissions or emergency visits forcardiovascular reasons within Day 30 episodes of worsening HF defined bythe need to increase the dose or reinitiate i.v. therapy with diureticsand/or other inotropic agents during the hospitalization will besummarized by treatment groups using descriptive statistics. Length ofhospitalization will be summarized by treatment group using descriptivestatistics. Safety endpoints The number and the percentage of patientsexperiencing adverse events, adverse drug reactions, serious adverseevents and adverse events leading to study withdrawal will be summarizedby treatment group. Adverse events will also be summarized by treatmentgroup by means of System Organ Class and Preferred Term using the MedDRAdictionary. Vital signs (including body temperature and dyspnoea),12-lead ECG parameters, incidence of clinically or hemodynamicallysignificant episodes of supraventricular or ventricular arrhythmias,laboratory parameters, renal function, cTNT, increase of cTNT andmortality will be summarized by treatment group using descriptivestatistics.

Echocardiography was performed on patients according to internationalstandards (see, for example, Lang R M et al., J Am Soc Echocardiogr2005; 18(12):1440-63; Nagueh S F et al., Eur J Echocardiogr 2009;10(2):165-93; Evangelista A et al., Eur J Echocardiogr 2008;9(4):438-48). Echocardiography was performed by expert physicians orsonographers at the sites. Echocardiography was done at screening,baseline, 6 hours after infusion start, 24 hours after infusion start(just before the end of infusion), and 48 hours after infusion start.

The following parameters were recorded for each patient at eachtimepoint and centrally measured by the CoreLab:

1. Cardiac dimension measures:

-   -   a. Left ventricle end diastolic diameter (EDD): measured with        M-mode echocardiography at the level of mitral valve (MV)        leaflets from parasternal long axis view (PLAX) (normal range        [NR]: 42-59 mm males and 39-53 mm females);    -   b. Left ventricle end systolic diameter (ESD): measured with        M-mode echocardiography at the level of mitral valve (MV)        leaflets from PLAX (NR: 25-35 mm);    -   c. Left ventricle end diastolic volume (EDV): measured with        M-mode echocardiography at the level of mitral valve (MV)        leaflets from PLAX (NR: 67-155 mL males and 56-104 mL females);    -   d. Left ventricle end systolic volume (ESV): measured with        M-mode echocardiography at the level of mitral valve (MV)        leaflets from PLAX (NR: 22-58 mL males and 19-49 mL females);    -   e. Left atrium diameter (LAD): measured at end-ventricular        systole with M-mode echocardiography from PLAX. (NR: 30-40 mm        males and 27-38 mm females)    -   f. Left atrium area (LAA): measured from apical four chamber        view (NR: ≤20 cm²); and    -   g. Left atrium volume (LAV): derived from area-length measured        from apical four chamber view (NR: 18-58 mL males and 22-52 mL        females).

2. Left ventricle diastolic function parameters:

-   -   a. E wave: measured from mitral valve pulsed wave Doppler, is        the peak velocity of early filling. Normal range for all the        diastolic parameters significantly changes with age;    -   b. A wave: measured from mitral valve pulsed wave Doppler is the        peak velocity of late atrial filling. Not evaluable in patients        with AF;    -   c. E wave deceleration time (EDT): measured from mitral valve        pulsed wave Doppler represent the slope of the descending part        of E wave;    -   d. E/A ratio: determines the type of diastolic filling pattern        (normal E/A=1-2 and EDT=150-200 ms, abnormal relaxation E/A<1        and EDT≥240 ms, pseudonormal E/A=0.8-1.5; restrictive E/A≥2 and        EDT<160 ms). Not evaluable in patients with AF;    -   e. Ea: measured with tissue Doppler method at the lateral and        septal side of the mitral annulus from apical four chamber view        is the early diastolic velocity. The value has been calculated        as the average between Ea lateral and Ea septal. (NR≥10 cm/s)        (see Nagueh SF et al., Eur J Echocardiogr. 2009; 10(2):165-93);    -   f. Aa: measured with tissue Doppler method at the lateral and        septal side of the mitral annulus from apical four chamber view        is the late atrial diastolic velocity. The value has been        calculated as the average between Aa lateral and Aa septal. Not        evaluable in patients with AF; and    -   g. E/Ea ratio: this is a derived measure from E and Ea value.        This is highly correlated with left ventricle filling pressure        and with prognosis in patients with HF. (NR: <13) (see Nagueh S        F et al., supra).

3. Left ventricle systolic function parameters:

-   -   a. Left ventricle ejection fraction (LVEF): measured with        Simpson biplane method according to international        recommendations from apical four chamber view and apical two        chamber view. (NR≥55%) (see Lang R M et al., J Am Soc        Echocardiogr. 2005; 18(12):1440-63); and    -   b. Sa: measured with tissue Doppler method at the lateral and        septal side of the mitral annulus from apical four chamber view.        The value has been calculated as the average between Sa lateral        and Sa septal. Validation studies demonstrated that Sa        correlates with LVEF (NR≥6 cm/s) (see Gulati V K et al., Am J        Cardiol. 1996; 77(11):979-84).

4. Overall cardiac contraction parameters:

-   -   a. Stroke volume (SV): is a derived measure obtained with the        application of Bernoulli's formula using the dimension of left        ventricle outflow tract (LVOT) as diameter and LVOT time        velocity integral as velocity. (NR>60 mL/beat);    -   b. Cardiac output (CO): is derived by the multiplication of SV x        heart rate (HR) (NR: >4 L/min);    -   c. Stroke volume index (SVI): is a derived parameter obtained by        the adjustment of SV by body surface area (BSA) (NR: 33-47        mL/beat/m²); and    -   d. Cardiac index (CI): is a derived parameter obtained by the        adjustment of CO by body surface area (BSA) (NR: 2.5-4        L/min/m²).

5. Right ventricle function parameters:

-   -   a. Pulmonary arterial systolic pressure (PASP): estimated by the        sum of the peak velocity at tricuspidal continuous wave Doppler        and a fixed value derived from inferior vena cava diameter and        respiratory change. (NR<35 mmHg);    -   b. Tricuspid annular plane systolic excursion (TAPSE): measured        from M-mode echocardiography from apical four chamber view.        TAPSE correlates with right ventricle ejection fraction and its        reduction associated with worse prognosis in HF. (NR>16 mm) (see        Ghio S et al., J Am Coll Cardiol 2001; 37(1):183-8); and    -   c. Right ventricle Sa: measured with tissue Doppler method at        right ventricle free wall from apical four chamber view. Sa is a        derived parameter of systolic function and correlated with right        ventricle ejection fraction. (NR>10 cm/s) (see Voelkel N F et        al., Circulation 2006; 114(17):1883-91; Haddad F et al.,        Circulation 2008; 117(13):1717-31).

6. Other parameters:

-   -   a. Mitral regurgitation (MR): evaluated with a visual        qualitative assessment ang graded in four categories: none,        mild, moderate, and severe (see Lancellotti P et al., Eur J        Echocardiogr 2010; 11(4):307-32); and    -   b. Inferior vena cava diameter (IVC): measured with M-mode        echocardiography from subcostal view at 1-2 cm from the junction        with right atrium. This parameter has been used to estimate        systolic pulmonary artery pressure. It correlated with right        atrium pressure indicating the grade of congestion. Increased        IVC diameter is associated with prognosis in patients with HF        (NR: ≤1.5 cm) (see Pellicori P et al., JACC Cardiovasc Imaging        2013; 6(1):16-28; Voelkel N F et al., Circulation 2006;        114(17):1883-91).

TABLE 1A Cardiac changes at 6 hours of infusion. Ista 0.5 Ista 1.0Parameter μg/kg/min μg/kg/min Placebo p Ista 0.5 p Ista 1.0 LAA (cm²)−0.33 ± 1.885 −0.84 ± 2.421 −0.52 ± 1.840 0.663 0.521 LAV (ml)  −1.16 ±11.265  −4.92 ± 14.390  −3.24 ± 10.047 0.399 0.558 Diastolic function Ewave (cm/s)  −3.33 ± 14.764  −9.13 ± 17.464  −1.10 ± 10.990 0.451 0.018A wave (cm/s)  1.76 ± 13.572  5.56 ± 17.280  1.55 ± 10.368 0.955 0.348EDT (ms)  6.83 ± 47.921  20.37 ± 50.849  −0.18 ± 38.939 0.491 0.052 E/Aratio −0.286 ± 0.866  −0.317 ± 0.898  −0.124 ± 0.866  0.566 0.458 e’(cm/s)  0.61 ± 1.010  0.01 ± 1.154  0.25 ± 1.167 0.146 0.376 E/e’ ratio−3.183 ± 5.628  −2.028 ± 3.652  −0.740 ± 3.994  0.032 0.150 Systolicfunction Sa (cm/s) Left V 0.613 ± 1.035 0.908 ± 0.936 0.197 ± 0.9190.065 0.001 S (cm/s) Right V  1.25 ± 2.185  2.00 ± 1.907  0.43 ± 1.4410.125 0.003 Cardiac function CO (l/min) 0.385 ± 0.843 0.228 ± 0.7600.083 ± 0.705 0.094 0.390 CI (l/min/m2) 0.209 ± 0.445 0.140 ± 0.4340.042 ± 0.400 0.090 0.309 SV (ml/beat)  7.724 ± 11.752 7.269 ± 8.1342.405 ± 7.244 0.020 0.007 SVI (ml/beat/m2) 4.198 ± 6.218 4.187 ± 4.6411.317 ± 4.077 0.019 0.005

TABLE 1B Cardiac changes at 24 hours of infusion. Ista 0.5 Ista 1.0Parameter μg/kg/min μg/kg/min Placebo p Ista 0.5 p Ista 1.0 LAA (cm²)−1.70 ± 2.463 −2.56 ± 2.972 −0.31 ± 1.886 0.008 <0.001  LAV (ml)  −7.94± 13.269 −13.81 ± 17.198  −2.95 ± 10.624 0.079 0.002 Diastolic functionE wave (cm/s)  −8.14 ± 17.640 −14.24 ± 24.416  −4.16 ± 12.502 0.2670.031 A wave (cm/s)  5.13 ± 10.990  11.10 ± 16.498 −1.14 ± 7.580 0.0450.003 EDT (ms)  12.88 ± 54.954  9.79 ± 52.212  4.58 ± 35.759 0.456 0.628E/A ratio −0.647 ± 0.812  −0.722 ± 1.068  −0.164 ± 0.833  0.005 0.004 e’(cm/s)  0.94 ± 1.089  0.91 ± 1.792 0.26 ± 1.23 0.013 0.635 E/e’ ratio−4.548 ± 4.754  −3.191 ± 2.623  −1.285 ± 3.351  0.001 0.011 Systolicfunction Sa (cm/s) Left V 0.679 ± 0.907 0.803 ± 1.023 0.171 ± 1.0290.024 0.012 S (cm/s) Right V  1.10 ± 1.780  1.55 ± 2.012  0.15 ± 1.4240.052 0.015 Cardiac function CO (l/min) 0.486 ± 0.696 0.239 ± 0.7860.195 ± 0.651 0.073 0.797 CI (l/min/m²) 0.264 ± 0.369 0.140 ± 0.4430.116 ± 0.373 0.097 0.808 SV (ml/beat)  9.725 ± 12.192 9.339 ± 8.8754.039 ± 6.259 0.015 0.005 SVI (ml/beat/m²) 5.333 ± 6.664 5.318 ± 4.9552.336 ± 3.516 0.019 0.005

TABLE 1C Cardiac changes a 48 hours of infusion. Ista 0.5 Ista 1.0Parameter μg/kg/min μg/kg/min Placebo p Ista 0.5 p Ista 1.0 Diastolicfunction E/A ratio −0.278 ± 1.043  −0.356 ± 0.967  −0.172 ± 0.741  0.7290.509 E/e’ ratio −2.570 ± 3.654  −2.218 ± 3.193  −1.800 ± 4.013  0.3880.629 Systolic function LVEF (%)  1.28 ± 3.693  2.06 ± 4.975  1.24 ±3.539 0.968 0.427 Sa (cm/s) 0.224 ± 0.811 0.257 ± 0.771 0.132 ± 0.8980.640 0.525 Cardiac function CO (l/min) 0.350 ± 0.788 0.368 ± 0.735−0.017 ± 0.691  0.041 0.026 CI (l/min/m²) 0.191 ± 0.426 0.205 ± 0.407−0.007 ± 0.384  0.044 0.027 SV (ml/beat) 4.815 ± 9.832 5.203 ± 8.0781.787 ± 7.108 0.139 0.062 SVI (ml/beat/m²) 2.762 ± 5.603 2.913 ± 4.5721.039 ± 4.026 0.139 0.071

The quantitative determination of PST 2744 and its metabolite PST 2915in human plasma was determined by the HPLC-MS/MS method that included amobile phase of 70:30 acetonitrile/water, 1 mL/L 1M formic acid, and 1mL/L 5M ammonium acetate. The flow rate was 1 mL/min, and thechromatographic separation was by reversed phase HPLC (Column: SYNERGI4μ POLAR-RP80A 150×4.6 mm equipped with a Security-guard PhenomenexPolar-RP 4×3 mm). Detection was performed by MS/MS, and the acquisitionmode was Multiple Reaction Monitoring (MRM).

The quantitative determination of istaroxime metabolies PST 2922 and PST3093 in human plasma was also measured by the HPLC-MS/MS method. In thiscase, the mobile phase was 50:50 H2O/CH3CN (v/v) and 500 μL/L 98-100%HCOOH. The flow rate was 1 mL/min and chromatographic separation wasdone by reversed-phase HPLC (Column: Phenomenex Phenyl hexyl, 150×4.6mm, equipped with a Phenomenex phenyl propyl guard-cartridge) underisocratic conditions. Detection was performed by by MS/MS (376.0→282.0amu for PST2922, 378.0→284.0 amu for PST 3093 and 362.0→268.0 amu forPST 3418, IS).

The data reported in Table 1A clearly indicates that infusion at either0.5 μg/kg/min or 1 μg/kg/min istaroxime does not significantly improvemost of the altered echo parameters of diastolic function (E, A waves,E/A ratio), with significant reduction occurring only with E/e′ at 0.5μg/kg/min. The systolic function (Sa and S wave) is improved at 1μg/kg/min.

Surprisingly, when istaroxime was infused for 24 hours, a clear andstatistically significant improvement is observed for most of thediastolic function parameters (E and A waves, E/A and E/e′ ratios),while the positive effect of systolic function (Sa and S wave) ismaintained but does not continue to increase (see Table 1B). At 48hours, both the CO and CI changes are still significantly increased overplacebo (see Table 1C). The shift from increased changes of SV and SVIat 6 and 24 hours to changes in CO and CI at 48 hours is also favouredby the normalization of the decreased HR at 6 and 24 hours, which wasdemonstrated in the previous Horizon study and confirmed in the presentstudy. The changes of the other indexes of cardiac relaxation andcontraction are still present, but do not achieve the statisticalsignificance.

FIGS. 2A-2C show the plasma concentrations of istaroxime and itsmetabolites in Caucasian and Chinese patients during and subsequent toinfusion with 0.5 or 1 μg/kg/min of istaroxime for 24 hours. During theinfusion period, both istaroxime (2744) and istaroxime metabolite PST2922 remain relatively constant and are rapidly cleared after infusionis stopped. On the other hand, istaroxime metabolites PST 2915 and PST3093 continue to accumulate in the plasma throughout the infusion periodwith the average concentration of PST 3093 exceeding 60 ng/mL by the endof the infusion period and with average concentration levels exceeding10 ng/mL even at 70 hours, or 46 hours post-infusion.

Notably, at 48 hours of infusion, the plasma levels of istaroxime arenot detectable after 20 hours, while those of metabolite PST 3093average about 25.02 ng/ml in Caucasian patients after 0.5 μg/kg/mlistaroxime infusion, about 12.5 ng/ml in Chinese patients after 0.5 μcgμg/kg/ml istaroxime infusion, and about 21.2 ng/ml with 1 μg/kg/mlistaroxime infusion (see FIGS. 2A-2C). These concentrations are muchhigher than the concentrations of 3093 exhibiting SERCA2a-stimulatoryactivity in SR vesicles from normal canine heart as shown in Table 3.Moreover, according to Ferrandi M et al. (BJP 2013; 169:1849-1861),istaroxime exerts its maximum SERCA2a activation in SR vesicles fromfailing canine hearts at concentrations that are much lower (about 10times) than those able to stimulate this activity in SR vesicles fromhealthy canine heart. Therefore, it is likely that this remarkabledifference between normal and failing heart SR vesicles may also occurfor PST 3093, which may continue to maintain its SERCA2a stimulatoryactivity even at the lower concentrations detected at 72 hours (seeFIGS. 2A-2C), where no echocardiographic data are available.

While not intending to be bound by theory, the above-discussedobservation is consistent with the hypothesis that a pure SERCA2aactivator may improve cardiac pump function. Finally this change inefficacy of cardiac pump function is not associated with any significantchanges in plasma level of Hs TnT, which is considered by cardiologistsas the most reliable biomarker of myocardial damage. This lack of Hs TnTchange is likely due the activation of SERCA2a that, by reducing thecardiomyocytes plasma Ca²⁺ concentration, also minimizes thecardiomyocytes damage. At present, the stimulation of the cardiacpumping ability by the only available inotropic agent under development(omecamtiv) is associated with an increase in the plasma levels of HsTnT, and different developmental strategies are under study to detectthe dose that minimize these changes in plasma HsTnT (Teerlink J R etal., 2016 Lancet 388, 2895-903).

The present invention may provide the basis for planning appropriatetrials aimed at assessing whether different ratios between plasma levelsof PST 3093 and istaroxime, achievable by varying the dose and durationof istaroxime infusion, may furnish greater therapeutic benefits topatients with HFpEF or HFmEF than to patients with HFrEF, or in patientswith or without Echocardiographic indexes revealing a status ofdiastolic impairment; thus increasing the precision of the therapeuticapproach to AHF.

Pharmaceutical Compositions

Pharmaceutical compositions and formulations for intravenous infusioncomprising istaroxime or a metabolite thereof in admixture with at leastone conventional pharmaceutically acceptable carrier and/or vehicleand/or excipient are commonly known in the art.

The pharmaceutical compositions and formulations for intravenousinfusion can be formulated in any way and can be administered in avariety of unit dosage forms depending upon the condition or disease andthe degree of illness, the general medical condition of each patient,the resulting preferred method of administration and the like. Detailson techniques for formulation and administration are well described inthe scientific and patent literature, see, e.g., the latest edition ofRemington's Pharmaceutical Sciences, Mack Publishing Co, Easton Pa.(“Remington's”).

The formulations may conveniently be presented in unit dosage form andmay be prepared by any method known in the art of pharmacy. The amountof active ingredient which can be combined with a carrier or vehiclematerial to produce a single dosage form will vary depending upon thesubject being treated and the particular mode of administration. Theamount of active ingredient that can be combined with a carrier materialto produce a single dosage form will generally be the amount of thecompound which produces a therapeutic effect.

Pharmaceutical formulations as provided herein can be prepared accordingto any method known to the art for the manufacture of pharmaceuticals.Such formulations can contain additional agents, such as preserving orstabilizing agents. A formulation can be admixtured with nontoxicpharmaceutically acceptable carriers or excipients which are suitablefor manufacture. Formulations may comprise one or more diluents,emulsifiers, preservatives, buffers, excipients, etc. and may beprovided in such forms as liquids, powders, emulsions, lyophilizedpowders, etc.

Aqueous suspensions can contain an active agent (e.g., a compositionused to practice the uses and methods as provided herein) in admixturewith excipients suitable for the manufacture of aqueous suspensions.Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethylene oxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol (e.g.,polyoxyethylene sorbitol mono-oleate), or a condensation product ofethylene oxide with a partial ester derived from fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). Theaqueous suspension can also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate. Formulations can be adjusted forosmolarity.

According to the present invention, istaroxime is given by intravenous(IV) administration. These formulations can comprise a solution ofactive agent dissolved in a pharmaceutically acceptable carrier.Acceptable vehicles and solvents that can be employed are water,dextrose in water, and Ringer's solution, an isotonic sodium chloride.These solutions are sterile and generally free of undesirable matter.These formulations may be sterilized by conventional, well knownsterilization techniques. The formulations may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents, e.g., sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate and the like. The concentration ofactive agent in these formulations can vary widely, and will be selectedprimarily based on fluid volumes, viscosities, body weight, and thelike, in accordance with the particular mode of administration selectedand the patient's needs. The administration is by bolus or continuousinfusion (e.g., substantially uninterrupted introduction into a bloodvessel for a specified period of time).

Istaroxime as provided herein can be lyophilized. Provided herein is astable lyophilized formulation comprising a composition as providedherein, which can be made by lyophilizing a solution comprising apharmaceutical as provided herein and a bulking agent, e.g., mannitol,trehalose, raffinose, and sucrose or mixtures thereof. There are manyother conventional lyophilizing agents. Among the sugars, lactose is themost common. Also used are citric acid, sodium carbonate, EDTA, Benzylalcohol, glycine, sodium chloride, etc. (see, for example, Journal ofExcipients and Food Chemistry Vol. 1, Issue 1 (2010) pp 41-54; U.S.patent app. no. 20040028670). In a preferred embodiment, istaroxime canbe prepared as powder for injection according to the teaching ofCN103315968.

According to the present invention, istaroxime as provided herein can beadministered for prophylactic and/or therapeutic treatments. Intherapeutic applications, compositions are administered to a subjectalready suffering from a condition, or disease in an amount sufficientto treat, prevent, cure, alleviate or partially arrest the clinicalmanifestations of the condition, or disease and its complications (i.e.,a “therapeutically effective amount”). For example, in alternativeembodiments, pharmaceutical compositions as provided herein areadministered in an amount sufficient to treat, prevent or ameliorate inan individual in need thereof. The amount of pharmaceutical compositionadequate to accomplish this is defined as a “therapeutically effectivedose.” The dosage schedule and amounts effective for this use, i.e., the“dosing regimen,” will depend upon a variety of factors, including thestage of the disease or condition, the severity of the disease orcondition, the general state of the patient's health, the patient'sphysical status, age and the like. In calculating the dosage regimen fora patient, the mode of administration also is taken into consideration.

The dosage regimen also takes into consideration pharmacokineticsparameters well known in the art, i.e., the active agents'bioavailability, metabolism, clearance, and the like (see, e.g.,Hidalgo-Aragones J., Steroid Biochem. Mol. Biol. 1996; 58:611-617;Groning, Pharmazie 1996; 51:337-341; Fotherby Contraception 1996;54:59-69; Johnson, J. Pharm. Sci. 1995; 84:1144-1146; Rohatagi,Pharmazie 1995; 50:610-613; Brophy, Eur. J. Clin. Pharmacol. 1983;24:103-108; the latest Remington's, supra). The state of the art allowsthe clinician to determine the dosage regimen for each individualpatient, active agent and disease or condition treated. Guidelinesprovided for similar compositions used as pharmaceuticals can be used asguidance to determine the dosage regimen, i.e., dose schedule and dosagelevels, administered practicing the methods as provided herein arecorrect and appropriate.

Single or multiple administrations of formulations can be givendepending on the dosage and frequency as required by the AHF clinicalsymptoms of patient. The formulations should provide a sufficientquantity of active agent to effectively treat or prevent or ameliorate aconditions, diseases or symptoms as described herein. A correcttreatment of AHF, by selectively normalizing a depressed biochemicalactivity underlying the symptoms of subset of patients (HFpEF or HFmEF),may be expected to selectively improve the symptoms and to reduce theincidence of unwanted side effects produced by the available drugseither during hospital staying or after discharge. The term preventionis applicable when the continuous monitoring of the pulmonary pressureis possible with the appropriate chronic implantable devices thatfurnish and estimation of PCWP. In this condition a significant increasein the PCWP may precede the appearance of symptoms of AHF, thusproviding the rational to infuse the Istaroxime at the right dose toprevent the symptoms and the consequent hospitalization

In one embodiment, an effective amount of istaroxime or an equivalent ofa pharmaceutically acceptable salt, solvate or hydrate thereof,administered to an individual in need thereof comprises use of variousdosing schedules, e.g.: from about 0.1 μg/kg/min to about 3.0 μg/kg/min,e.g., 0.1 μg/kg/min, 0.15 μg/kg/min, 0.2 μg/kg/min, 0.25 μg/kg/min, 0.3μg/kg/min, 0.35 μg/kg/min, 0.4 μg/kg/min, 0.5 μg/kg/min, 0.6 μg/kg/min,0.7 μg/kg/min, 0.8 μg/kg/min, 0.9 μg/kg/min, 1.0 μg/kg/min, 1.1μg/kg/min, 1.2 μg/kg/min, 1.3 μg/kg/min, 1.4 μg/kg/min, 1.5 μg/kg/min,1.6 μg/kg/min, 1.7 μg/kg/min, 1.8 μg/kg/min, 1.9 μg/kg/min, 2.0μg/kg/min, 2.1 μg/kg/min, 2.2 μg/kg/min, 2.3 μg/kg/min, 2.4 μg/kg/min,2.5 μg/kg/min, 2.6 μg/kg/min, 2.7 μg/kg/min, 2.8 μg/kg/min, 2.9μg/kg/min, or 3.0 μg/kg/min. For instance, in some embodiments,istaroxime or its metabolite (e.g., PST 3093), is administered byinfusion at an effective dose from about 0.2 μg/kg/min to about 2.0μg/kg/min, or from about 0.2 μg/kg/min to about 1.5 μg/kg/min, or fromabout 0.25 μg/kg/min to about 1.0 μg/kg/min, or from about 0.5 μg/kg/minto about 1.0 μg/kg/min.

In alternative embodiments, an effective amount of istaroxime or anequivalent of a pharmaceutically acceptable salt, solvate or hydratethereof, administered to an individual in need thereof is individualizedbased on basal levels and subsequent changes of certain heart functionparameters, such as echo indexes or Pulmonary Capillary Wedge Pressure(PCWP), dyspnea, peripheral and pulmonary venous congestion, urinaryvolume, serum biomarkers such as NT-proBNP and high sensitive cardiacTroponin (hs-cTnT).

In alternative embodiments, an effective amount is demonstrated byreduction of PCWP, orthopnea, paroxysmal nocturnal dyspnea, reduction ofperipheral and pulmonary venous congestion, such as pulmonarycrepitations or rales, reduction of ankle swelling, reduction ofbiomarkers urinary output such as NT-proBNP and high sensitive cardiacTroponin (hs-cTnT).

In alternative embodiments, an effective amount of istaroxime or anequivalent of a pharmaceutically acceptable salt, solvate or hydratethereof, administered to an individual in need thereof is individualizedbased on basal levels and subsequent changes of certain heart functionparameters, such as echo indexes or Pulmonary Capillary Wedge Pressure(PCWP), dyspnea, peripheral and pulmonary venous congestion, urinaryvolume, serum biomarkers such as NT-proBNP and high sensitive cardiacTroponin (hs-cTnT).

In alternative embodiments, an effective amount is demonstrated byreduction of PCWP, orthopnea, paroxysmal nocturnal dyspnea, reduction ofperipheral and pulmonary venous congestion, such as pulmonarycrepitations or rales, reduction of ankle swelling, reduction ofbiomarkers urinary output such as NT-proBNP and high sensitive cardiacTroponin (hs-cTnT).

Methods of Treatment

Also provided herein are methods of treating an individual with heartfailure. In preferred embodiments, the individual exhibits symptoms of,or has been diagnosed with, acute heart failure. While the individualcan be a non-human animal, in a preferred embodiment, the individual isa human patient, such as a human patient suffering from heart failure.

In general, the compositions described herein can be used to treat theindividual having heart failure or acute heart failure. In anembodiment, the method of therapy includes providing or presenting theindividual having heart failure or acute heart failure. In some cases, ameasuring step is first carried out to determine the baseline heartfunction of the individual. For instance, an individual with heartfailure may exhibit impaired or decreased diastolic relaxation function.The measuring step may include measuring one or more parameters of heartfailure, such as, but not limited to, decreased heart rate, decreasedheart pressure, decreased systolic and/or diastolic blood pressure,reduced left ventricular end-diastolic/systolic volume and function(LVEF), or increased E/Ea or E/A ratios reduced Ea ratio decreasedstroke volume. As one having ordinary skill in the art will appreciate,any suitable measuring technique available in the art at the time of themeasuring step is suitable for use herein, and it is well within thepurview of such skilled artisan to select an appropriate measuringtechnique corresponding to the parameter of interest. A non-limitinglist of suitable measuring equipment/techniques includes echocardiogram,cardiac catheterization, nuclear stress test, CAT scan, radionuclideventriculography scan, stethoscope, sphygmomanometer, and the like. Forinstance, the diastolic relaxation can be measured by echocardiographyor PCWP.

The methods disclosed herein also include administering to theindividual a therapeutically effective amount of istaroxime or ametabolite thereof, such as PST 3093. In preferred embodiments, theistaroxime or istaroxime metabolite is in a pharmaceutical composition,such as any one of the combinations discussed above. The istaroxime oristaroxime metabolite is administered in an therapeutically effectivedose as disclosed elsewhere herein, e.g., between about 0.25 μg/kg/minto about 1.0 μg/kg/min. In a more preferred embodiment, the route ofadministration is infusion, such as intravenous fusion. The measuringstep can be performed before, during, or after the administering step.For instance, it may be desired to continually monitor one or more ofthe parameters of heart function during treatment and for a period oftime thereafter.

As discussed above, it has been surprisingly discovered thatadministering istaroxime (or its metabolites) by infusion for aninfusion duration of greater than 6 hours, e.g., 6.1 h, 6.2 h, 6.3 h,6.4 h, 6.5 h, 6.6 h, 6.7 h, 6.8 h, 6.9 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36h, 37 h, 38 h, 39 h, 40 h, 41 h, 42 h, 43 h, 44 h, 45 h, 46 h, 47 h, or48 h or more, results in increased SERCA2a activity and improvingdiastolic relaxation without causing arrhythmogenic effects due to, forexample, Na⁺/K⁺ pump inhibition. In this manner, istaroxime infusion forgreater than 6 hours exerts a lusitropic-SERCA2a activity that isprevailing on the inotropic activity and results in improved diastolicrelaxation as compared to istaroxime infusion for less than 6 hours.

While not intending to be bound by theory, it is believed that thislater-arising “pure” SERCA2a activation is due to an accumulation ofistaroxime metabolites in the plasma of the individual. As such, in someembodiments, istaroxime is administered via intravenous infusion for aperiod of time sufficient to enable the accumulation of istaroximemetabolites in the plasma of the individual. In preferred embodiments,the infusion duration is sufficient to allow for the accumulation of oneor more istaroxime metabolites; preferably, the metabolite is PST 2915having the structural formula (II) or PST 3093 having the structuralformula (III); more preferably, the metabolite is PST 3093. In someembodiments, the accumulation of istaroxime metabolite in the plasma isat a concentration of least about 3 ng/mL, e.g., 3 ng/mL, 4 ng/mL, 5ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, 10 ng/mL, 11 ng/mL, 12 ng/mL,13 ng/mL, 14 ng/mL, 15 ng/mL, 16 ng/mL, 17 ng/mL, 18 ng/mL, 19 ng/mL, 20ng/mL, 21 ng/mL, 22 ng/mL, 23 ng/mL, 24 ng/mL, 25 ng/mL, 26 ng/mL, 27ng/mL, 28 ng/mL, 29 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50ng/mL or more for a period of time of at least about 3 hours, e.g., 3 h,4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h,17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h or more. In oneembodiment, the istaroxime metabolite accumulates in the plasma to thedesired concentration within 6 hours; preferably, within 3 hours orwithin 2 hours or within 1 hour of istaroxime infusion initiation and ismaintained at or above that concentration for at least about 3additional hours, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or more additional hours; preferably,for at least 6 additional hours; more preferably for at least about 12additional hours. In some embodiments, the desired plasma concentrationof metabolite is at least about 5 ng/mL. In another embodiment, theistaroxime metabolite accumulates in the plasma to a concentration of atleast about 10 ng/mL and is maintained at or above that concentrationfor at least about 6 additional hours; preferably for at least about 12additional hours. In yet another embodiment, the istaroxime metaboliteaccumulates in the plasma to a concentration of at least about 15 ng/mLand is maintained at or above that concentration for at least about 6additional hours; preferably for at least about 12 additional hours. Insome embodiments, the plasma concentration of the metabolite remainsabove 5 ng/mL for at least about 6 hours following the completion of theistaroxime administration. In some embodiments, the plasma concentrationof the metabolite remains above 10 ng/mL for at least about 6 hoursfollowing the completion of the istaroxime administration. In yet otherembodiments, the plasma concentration of the metabolite remains above 20ng/mL for at least about 6 hours following the completion of theistaroxime administration. In others, the concentration remains at leastabout 30 ng/mL, 40 ng/mL, or 50 ng/mL for an additional 6 hours or more,e.g., 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17h, 18 h, 19, 20 h, 21 h, 22 h, 23 h, 24 h or more following istaroximeinfusion. For instance, the metabolite accumulation may remain at aconcentration level of at least about 10 ng/mL for an additional 12hours or more, e.g., 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19, 20 h,21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h,33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h, 40 h, or more following theistaroxime infusion.

As one having ordinary skill in the art would appreciate, the plasmaconcentration of istaroxime or istaroxime metabolite can be measured byconventional means, such as by HPLC-MS/MS.

In some embodiments, the istaroxime metabolites have the structuralformulas (II) or (III):

In preferred embodiments, the metabolite of istaroxime is the compoundPST 3093.

In a further example, an individual is diagnosed with heart failure oracute heart failure, and is administered about 0.25 μg/kg/min to about1.0 μg/kg/min of istaroxime for a period of time that is greater than 6hours. As the istaroxime is metabolized by the individual, istaroximemetabolites, such as PST 3093, begin to accumulate in the plasma of theindividual. For instance, istaroxime metabolite PST 3093 may accumulateto a plasma concentration of at least about 5 ng/mL within 3 hours ofistaroxime infusion and is maintained at a plasma level of at leastabout 5 ng/mL for the duration of the istaroxime infusion and for anadditional 6 to about 36 hours. The presence of the PST 3093 acting as a“pure” SERCA2a activator confers to the individual improved diastolicrelaxation.

In some embodiments, the method of treatment may include administrationof the istaroxime metabolite by infusion in combination with or insteadof istaroxime. For instance, a method of treating an individual withheart failure is disclosed that includes administering to the individuala therapeutically effective amount of a pharmaceutical composition thatcomprises a pharmaceutically acceptable carrier and an istaroximemetabolite having the formula (II) or (III). In preferred embodiments,PST 3093 or PST 2915 is the metabolite; more preferably, it is PST 3093.Such methods may include a measuring step, wherein one or moreparameters of heart function are measured using measuring techniquesavailable in the art. The measuring step may be performed prior to,during, or subsequent to the administration of the pharmaceuticalcomposition. In such methods, the therapeutically effective dose of PST3093 will be between about 0.2 μg/kg/min to about 2.0 μg/kg/min;preferably between about 0.3 μg/kg/min and about 1.5 μg/kg/min; morepreferably between about 0.5 μg/kg/min and about 1.0 μg/kg/min. In suchmethods, the duration of infusion will be greater than 6 hours, e.g.,6.1 h, 6.2 h, 6.3 h, 6.4 h, 6.5 h, 6.6 h, 6.7 h, 6.8 h, 6.9 h, 7 h, 8 h,9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h,21 h, 22 h, 23 h, 24 h, 36 h, 48 h, or more.

In some embodiments, it is desired to treat an individual with acuteheart failure with two or more istaroxime dose regimens to initiallytreat the acute heart failure symptoms and subsequently normalize theheart functional defects associated with a more chronic heart failurecondition. In such embodiments, the dosing regimen is manipulated tocontrol the istaroxime to metabolite ratio by starting with theadministration by infusion of a higher dose of istaroxime to treat acuteheart failure symptoms followed by administering a lower istaroxime doseby infusion for a longer duration to allow for the accumulation ofistaroxime metabolites. In this manner, the initial infusion will allowfor rapid SERCA2a stimulation and Na,K-ATPase inhibition resulting in arapid positive inotropy to treat the acute heart failure symptoms, whilethe second infusion will allow for accumulation of the selectiveSERCA2a-activating istaroxime metabolite PST 3093 and extended positiveinotropy at a reduced risk of arrhythmogenic Ca²⁺ triggering events.

Therefore, provided herein is a method of treating an individual havingacute heart failure, wherein it is administered to the individual byinfusion a first pharmaceutical composition that includes apharmaceutically acceptable carrier and istaroxime at a therapeutic doseof at least about 1.5 μg/kg/min. The heart function of the individualcan be measured and monitored before, during, and/or after beginninginfusion of the first pharmaceutical composition using any of thetechniques discussed herein. For instance, in some embodiments, one ormore parameters of heart function are measured prior to administeringistaroxime in order to determine, e.g., whether the individual ispresenting with HFpEF or HFmEF. In other embodiments, initialmeasurements may include echocardiogram or PCWP values to measurediastolic relaxation dysfunction. These measurements may also beinitiated concurrently with administering the istaroxime by infusionand/or may be continued throughout the duration of the infusion. In someembodiments, the step of measuring one or more parameters of heartfunction can be performed after administering the first pharmaceuticalcomposition.

In the dosing manipulation methods, once an improvement in theparameters of heart function are measured, a pharmaceutical compositionthat comprises a pharmaceutically acceptable carrier and istaroxime at alower therapeutic dose is administered by infusion. For instance, animprovement in diastolic relaxation in the individual as measured byechocardiogram or PCWP as compared to the same measurements taken priorto and/or at the start of the initial infusion would indicate treatmentof the acute heart failure symptoms. Also, the reduction of acute heartfailure symptoms such as breathlessness, ankle swelling, elevatedjugular venous pressure, pulmonary crackles and peripheral edema mayjustify the change in the infusion rate. In some embodiments, the secondtherapeutic dose of istaroxime is less than about 1.5 μg/kg/min;preferably between about 0.3 μg/kg/min and about 1.5 μg/kg/min; morepreferably between about 0.5 μg/kg/min and about 1.0 μg/kg/min. Forinstance, in one particular embodiment, the second therapeutic dose ofistaroxime is about 0.5 μg/kg/mim. Administering by infusion of thesecond therapeutic dose of istaroxime may then be continued foraduration of greater than 6 hours, e.g., 6.1 h, 6.2 h, 6.3 h, 6.4 h, 6.5h, 6.6 h, 6.7 h, 6.8 h, 6.9 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 36 h, 48h, or more.

In some embodiments, the second, lower dose of istaroxime may be infusedfor a duration sufficient to produce an accumulated plasma concentrationof an istaroxime metabolite, such as PST 3093. In such embodiments,infusion of the lower dose of istaroxime is continued until the plasmaconcentration of PST 3093 is at least about 20 ng/mL. In otherembodiments, infusion of the lower dose of istaroxime is continued untilthe plasma concentration of PST 3093 is at least about 30 ng/mL. Instill other embodiments, infusion is continued until the plasmaconcentration of PST 3093 is at least about 40 ng/mL or at least about50 ng/mL or at least about 60 ng/mL. Once infusion of the lower dose ofistaroxime is stopped, the istaroxime compound is cleared from theindividual while the istaroxime metabolite exhibiting selective or“pure”SERCA2a activation (i.e., 3093) remains in the individual's bloodstreamfor extended periods of time to confer to the individual improved heartfunction with a much lower risk of arrhythmogenic triggering events.

In alternative embodiments, in evaluating the efficacy of a treatment, atreatment regimen or a particular dosage, or to determine if a treatmentversus a maintenance dosage should be given, individuals, e.g., patientsaffected by acute or chronic heart failure, are subject to regularperiodic screening for the presence and extent of organ and tissueinvolvement or damage, e.g., heart (ventricle dilatation, third heartsound cardiac hypertrophy), fatigue, tiredness, reduced exercisetolerance, increased time to recover after exercise, kidney (renalinsufficiency, oliguria), lung (orthopnea, paroxysmal nocturnal dyspnea,tachypnea), ankle swelling, elevated jugular venous pressure. A thoroughphysical examination should be done at a time interval chosen by thoseexperts in the treatment of a cardiovascular disease, in particularacute or chronic heart failure which would concentrate on cardiac,pulmonary and peripheral circulation functions. Accordingly, inalternative embodiments, therapy with istaroxime or an equivalent of apharmaceutically acceptable salt, solvate or hydrate thereof asdisclosed herein, is instituted as early as possible, preferably inemergency, to prevent the rapid evolution of symptoms and continuedafter patient's discharge for years, preferably during the whole life ofthe patient or at least a period consistent with the way other drugs areused in heart failure. As the result of monitoring of patient'sconditions by the medical doctor, istaroxime long infusion, longer than6 hours may be given the patient up to once/twice a month in order toprevent occurrence of acute episodes of heart failure, thus avoidingemergency rescue of the patient and lessening the probability oflife-threatening episodes.

According to the present invention, uses and methods as provided hereincan further comprise co-administration with other drugs orpharmaceuticals. In fact, the present invention selectively corrects adepressed cardiac biochemical function (namely the SERCA2a activity).This certainly contributes to relieving the existing HF clinicalsymptoms, with less unwanted side effects than those of the availabletherapies (just because the selectivity mentioned above). However, asCHF and AHF are complex clinical syndromes the present invention ispotentially associable to existing and future drug classes and/orspecific drugs such as: a) drug classes such as, ACE inhibitors, AIRBs,diuretics, Ca channel blockers, _(R) blockers, digitalis, NO donors,vasodilators, SERCA2a stimulators, neprilysin (NEP) inhibitors, myosinfilament activators, recombinant relaxin-2 mediators, recombinant NPprotein, activators of the soluble Guanylate Cyclase (sGC),beta-arrestin ligand of Angiotensin II receptor; b) specific drugs:hydrochlorothyzide, furosemide, verapamil, diltiazem, carvedilol,metoprolol, hydralazine, eplerenone, spironolactone, lisinopril,ramipril, nitroglycerin, nitrates, digoxin, valsartan, olmesartan,telmisartan, candesartan, losartan, entresto, omecamtiv, sacubitril,serelaxin, ularitide, levosimendan, cinaciguat. Subjects suffering fromheart failure treated with the above drugs and undergoing regularclinical monitoring, for example having their pulmonary blood pressurecontinuously monitored with implanted probes, can be guarded in order topredict episode of AHF that may be prevented by the infusion ofIstaroxime according to the present invention.

Istaroxime as disclosed in the present invention, as used a therapeuticagent for treating acute heart failure, can be combined with othertherapeutic agents used in the treatment of the same disease. Exemplaryother therapeutic agents are diuretics, for example furosemide,bumetanide, and torasemide. Metolazone, an aldosterone antagonist, suchas spironolactone or eplerenone; thiazide diuretics, such asHydrochlorothiazide, metolazone, and chlorthalidone. Other agents areACE inhibitors, for example Lisinopril and Ramipril. Also Angiotensin IIreceptor blockers (ARBs), such as valsartan, candesartan and losartancan be taken into consideration. Angiotensin receptor/neprilysininhibitor (ARNI), sacubitril for example, are comprised. Other agentscan be selected from Beta-blockers, such as carvedilol and metoprololfor example, or Vasodilators, for example Hydralazine, optionallycombined with isosorbide dinitrate, Nitrates, as nitroglycerin,amlodipine and felodipine; non-dihydropyridines such as diltiazem orverapamil. The compounds of the present invention can also be combinedwith Digoxin, if needed. Other drugs, as Ivabradine and otherAnticoagulant may be considered.

The compounds of the present invention can be combined with othertherapeutic agents, in particular agents useful for treatingcardiovascular diseases, more in particular in the combination therapyof heart failure. The combined active ingredients can be administeredaccording to different protocols, decided by the medical doctor.According to an embodiment of the present invention, combination therapycan be carried out by administering istaroxime both at the same time orat different time of the further therapeutically active ingredient oringredients. In case of concomitant administration, the compound of thepresent invention and the further active ingredient or ingredients canbe each formulated in a respective pharmaceutical composition or in thesame unitary dosage form. In the former case, the present inventionprovides a kit, in particular for the treatment of heart failure,comprising separate pharmaceutical compositions containing the compoundof the present invention and the further active ingredient oringredients, respectively. In another embodiment, the present inventionprovides a pharmaceutical unit dosage form kit, in particular for thetreatment of acute heart failure, comprising compound of the presentinvention and the further active ingredient or ingredients in the sameunit dosage form. Combination therapy according to the present inventionprovides advantageous treatment of heart failure due to theinotropic-lusitropic effect of istaroxime herein disclosed in additionto or synergically combined with the well-known therapeutic effect ofthe additional active agents herein disclosed.

Also provided are nanoparticles, nanolipoparticles, vesicles andliposomal membranes comprising compounds used to practice the uses andmethods as provided herein, e.g., to deliver pharmaceutically activecompounds and compositions as provided herein: istaroxime or a compoundof formula (II) or formula (III) or an equivalent of a pharmaceuticallyacceptable salt, solvate or hydrate thereof, optionally combined with afurther therapeutically active agent as disclosed above to a subject inneed thereof. In alternative embodiments, these compositions aredesigned to target specific molecules, including biologic molecules,such as polypeptides, including cell surface polypeptides, e.g., fortargeting a desired cell type, e.g., a myocyte or heart cell, anendothelial cell, and the like. A slow release of Istaroxime may providea sufficient compound to selectively increase the plasma levels of themetabolite leaving the plasma levels of Istaroxime within very lowranges.

Provided are multilayered liposomes comprising compounds used topractice methods as provided herein, e.g., as described in Park, et al.,U.S. application No. 20070082042. The multilayered liposomes can beprepared using a mixture of oil-phase components comprising squalane,sterols, ceramides, neutral lipids or oils, fatty acids and lecithins,to about 200 to 5000 nm in particle size, to entrap a composition usedto practice uses and methods as provided herein.

Liposomes can be made using any method, e.g., as described in U.S. Pat.No. 4,534,899; or Park, et al., U.S. application No. 20070042031,including method of producing a liposome by encapsulating an activeagent according to the present invention (or a combination of activeagents), the method comprising providing an aqueous solution in a firstreservoir; providing an organic lipid solution in a second reservoir,and then mixing the aqueous solution with the organic lipid solution ina first mixing region to produce a liposome solution, where the organiclipid solution mixes with the aqueous solution to substantiallyinstantaneously produce a liposome encapsulating the active agent; andimmediately then mixing the liposome solution with a buffer solution toproduce a diluted liposome solution.

In one embodiment, liposome compositions used to practice uses andmethods as provided herein comprise a substituted ammonium and/orpolyanions, e.g., for targeting delivery of istaroxime or an equivalentof a pharmaceutically acceptable salt, solvate or hydrate thereof usedto practice methods as provided herein to a desired cell type, asdescribed, e.g., in U.S. application No. 20070110798.

Provided are nanoparticles comprising compounds according to the presentinvention used to practice uses and methods as provided herein in theform of active agent-containing nanoparticles (e.g., a secondarynanoparticle), as described, e.g., in U.S. application No. 20070077286.In one embodiment, provided are nanoparticles comprising a fat-solubleactive agent used to practice a use and method as provided herein or afat-solubilized water-soluble active agent to act with a bivalent ortrivalent metal salt.

In one embodiment, solid lipid suspensions can be used to formulate andto deliver compositions used to practice uses and methods as providedherein to mammalian cells in vivo, in vitro or ex vivo, as described,e.g., in U.S. application No. 20050136121.

The compositions and formulations used to practice the uses and methodsas provided herein can be delivered by the use of liposomes ornanoliposomes. By using liposomes, particularly where the liposomesurface carries ligands specific for target cells, or are otherwisepreferentially directed to a specific organ, one can focus the deliveryof the active agent into target cells in vivo. See, e.g., U.S. Pat. Nos.6,063,400; 6,007,839; Al-Muhammed, J. Microencapsul. 1996; 13:293-306;Chonn Curr. Opin. Biotechnol. 1995; 6:698-708; Ostro, Am. J. Hosp.Pharm. 1989; 46:1576-1587. A liposome formulation of istaroxime asdisclosed in Eur J Pharm Biopharm. 2011; 79(2):285-93 is also providedin the present invention.

Delivery Vehicles

In alternative embodiments, any delivery vehicle can be used to practicethe uses and methods as provided herein, e.g., to deliver the compoundsprovided herein to a subject in need thereof. For example, deliveryvehicles comprising polycations, cationic polymers and/or cationicpeptides, such as polyethyleneimine derivatives, can be used e.g. asdescribed, e.g., in U.S. application No. 20060083737.

In one embodiment, a dried polypeptide-surfactant complex is used toformulate a composition used to practice a use and method as providedherein, e.g., as described in U.S. application No. 20040151766.

In one embodiment, a composition used to practice uses and methods asprovided herein can be applied to cells using vehicles with cellmembrane-permeant peptide conjugates, e.g., as described in U.S. Pat.Nos. 7,306,783; 6,589,503. In one aspect, the composition to bedelivered is conjugated to a cell membrane-permeant peptide. In oneembodiment, the composition to be delivered and/or the delivery vehicleare conjugated to a transport-mediating peptide, e.g., as described inU.S. Pat. No. 5,846,743, describing transport-mediating peptides thatare highly basic and bind to poly-phosphoinositides.

In one embodiment, electro-permeabilization is used as a primary oradjunctive means to deliver the composition to a cell, e.g., using anyelectroporation system as described e.g. in U.S. Pat. Nos. 7,109,034;6,261,815; 5,874,268.

The following examples further illustrate the present invention.

EXAMPLE 1 Preparation of the Compounds of Formula (II) and (III)Synthesis of PST 2915 Step 1: Hydroboration

To a solution of dehydroepiandrosterone I (30.0 g) in 450 mL of THFmaintained under a nitrogen atmosphere and at a temperature of −10° C.was added the complex BH.THF 1M in THF (260 mL). On completing theaddition, the temperature was allowed to rise once again to ambienttemperature; after 3 h 500 mL of H₂O were added and then NaBO₃.4H₂O(31.4 g). The reaction was left to stir for one night. The precipitateformed was filtered, washed with THF and eliminated. The aqueous andorganic phases were Separated, NaCl was added to the aqueous phase andthis was re-extracted with THF (3×200 mL). The combined organic phaseswere anhydrified with NaCl and Na₂SO₄ and evaporated under reducedpressure to obtain the crude product, which was crystallised byAcOEt/MeOH and then filtered and washed with AcOEt. Approximately 21 gof androstane 3β,6α,17β-triol II were obtained (known product:Nicholson, S. H., Turner, A. B. J. Chem. Soc., Perkin Trans. 1, 1976,1357 and U.S. Pat. No. 6,384,250 B2).

The analytical results are in agreement with those reported in theliterature.

Step 2: Selective Oxidation.

To a solution of Androstane 3β,6α,17β-triol II (30 grams) in a mixturecomposed by Dioxane (825 mL), Water (150 mL) and Pyridine (16.5 mL),N-Bromosuccinimide (52 grams) was added portion wise within 10 minutesprotecting the vessel from light. The mixture was stirred for 16 hoursat room temperature, diluted with 900 ml of water then Na₂S₂O₃ (15.5grams) were added portion wise within 15 minutes. The solution wasconcentrated (around 1500 mL were removed) and the suspension wasfiltered and the solid dried under vacuum giving 28.1 grams of6α-hydroxyandrostane-3,17-dione III (95% yield).

Step 3: Ketone Protection.

A suspension of 6α-hydroxyandrostane-3,17-dione III (18.85 grams) in 360mL of glycol and P-Toluenesulfonic Acid (554 mg) was heated at 100° C.and distilled under vacuum to remove the azeotropic mixture glycol/water(around 5 mL). The mixture was cooled and treated with 250 mg of KOHdissolved in 25 ml of Methanol. 15 mL of water were added and, afterstirring for 2 hours, the suspension was filtered giving intermediate IVas white solid (20.2 grams, 83% yield). The product was used withoutfurther purification.

Step 4: Oxidation.

A solution constituted by 3 mL of Sodium Hypochlorite (6%) and 28 mL ofEthyl Acetate were stirred at room temperature and 27 mg of RuO₂ hydratewere added. When all the catalyst Ruthenium was solubilized the productIV (1 gram) was added portion wise waiting for the disappearance ofblack suspension. After 1 hour additional 3 ml of Sodium Hypochlorite(6%) were added and the clear solution stirred for 3 hours at roomtemperature. When the reaction is completed the mixture was filtered ona Celite pad and the aqueous phase was extracted with AcOEt. Thecombined organic phases were washed with a solution of NaHCO₃ (5% inwater) and with NaCl (10% in water). The organic layer was dried overNa₂SO₄ and evaporated to dryness giving intermediate V (950 mg, 94%yield).

Step 5: Reduction.

A suspension of product V (5.76 grams) in Methanol (72 mL) was stirredat 0° C. and NaBH₄ (730 mg) was added. After 2 hours the reaction wascompleted, and the solvent was removed under reduced pressure. The crudeproduct was suspended in 30 mL of water and extracted with CH₂Cl₂. Theorganic layer was dried over Na₂SO₄ and evaporated to dryness. The crudesolid was purified by flash chromatography (SiO₂, Cyclohexane/AcOEt 7/3as eluent) giving the product VI (5.16 grams, 89% yield).

Step 6: Ketone Deprotection.

To a stirred solution of product VI (2.85 grams) in 350 mL of distilledAcetone, P-toluenesulfonic acid (7.14 grams) was added. After 3 hours atroom temperature a 5% solution of NaHCO₃ was added and the solvent wasremoved under reduced pressure. The product was extracted with CH₂Cl₂.The organic layer was dried over Na₂SO₄ and evaporated to dryness,giving the intermediate VII (2.13 grams, 95% yield).

Step 7: Synthesis of PST 2915.

To a stirred solution of 6β-hydroxyandrostane-3,17-dione VII (3.5 grams)in THF (100 mL), a solution of 2-aminoethoxyamine dihydrochloride (1.728grams) in H₂O (34 mL) was rapidly added dropwise. After 1.5 h at roomtemperature under vigorous stirring, NaCl (4 grams) was added and themixture stirred for 15 min. The phases were separated, and the aqueousphase was extracted twice with THF (2×50 mL). The combined organicextracts were dried over Na₂SO₄, filtered and evaporated to give a whitesolid (4.52 grams).

The crude product was suspended and slurried in 45 mL of AcOEt/EtOH 97/3for 1.5 hour then filtered and dried under reduce pressure at 35° C. for48 hours, giving(E,Z)-3-(2-Aminoethoxyimino)-6beta-hydroxyandrostan-17-onehydrochloride, PST2915 (4.069 grams, 89% yield).

Synthesis of PST 3093 Step 1: Hydroboration.

To a solution of dehydroepiandrosterone I (30.0 g) in 450 mL ofanhydrous THF maintained under a nitrogen atmosphere and at atemperature of −10° C. was added the complex BH.THF 1M in THF (260 mL).On completing the addition, the temperature was allowed to rise onceagain to room temperature; after 3 hours, 500 mL of H₂O were added andthen NaBO₃.4H₂O (31.4 g). The reaction was left to stir for one night.The precipitate formed was filtered, washed with THF and eliminated. Theaqueous and organic phases were separated, NaCl was added to the aqueousphase and this was re-extracted with THF (3×200 mL). The combinedorganic phases were anhydrified with NaCl and Na₂SO₄ and evaporatedunder reduced pressure to obtain the crude product, which wascrystallised by AcOEt/MeOH and then filtered and washed with AcOEt.Approximately 21 g of androstane 3β,6α,17β-triol II were obtained (knownproduct: Nicholson, S. H., Turner, A. B. J. Chem. Soc., Perkin Trans. 1,1976, 1357 and U.S. Pat. No. 6,384,250 B2).

The analytical results are in agreement with those reported in theliterature.

Step 2: Selective Oxidation

To a solution of Androstane 3β,6α,17β-triol II (30 grams) in a mixturecomposed by Dioxane (825 mL), Water (150 mL) and Pyridine (16.5 mL),N-Bromosuccinimide (52 grams) was added portion wise within 10 minutesprotecting the vessel from light. The mixture was stirred for 16 hoursat room temperature, diluted with 900 ml of water then Na₂S₂O₃ (15.5grams) were added portion wise within 15 minutes. The solution wasconcentrated (around 1500 mL were removed) and the suspension wasfiltered and the solid dried under vacuum giving 28.1 grams of6α-hydroxyandrostane-3,17-dione III (95% yield).

Step 3: Ketone Protection.

A suspension of 6α-hydroxyandrostane-3,17-dione III (18.85 grams) in 360mL of glycol and P-Toluenesulfonic Acid (554 mg) was heated at 100° C.and distilled under vacuum to remove the azeotropic mixture glycol/water(around 5 mL). The mixture was cooled and treated with 250 mg of KOHdissolved in 25 ml of Methanol. 15 mL of water were added and, afterstirring for 2 hours, the suspension was filtered giving intermediate IVas white solid (20.2 grams, 83% yield). The product was used withoutfurther purification.

Step 4: Oxidation.

A solution constituted by 3 mL of Sodium Hypochlorite (6%) and 28 mL ofEthyl Acetate were stirred and 27 mg of RuO₂ hydrate were added. Whenall the catalyst Ruthenium was solubilized the product IV (1 gram) wasadded portion wise waiting for the disappearance of black suspension.After 1 hour additional 3 ml of Sodium Hypochlorite (6%) were added andthe clear solution stirred for 3 hours at room temperature. When thereaction is completed the mixture was filtered on a Celite pad and theaqueous phase was extracted with AcOEt. The combined organic phases werewashed with a solution of NaHCO₃ (5% in water) and with NaCl (10% inwater). The organic layer was dried over Na₂SO₄ and evaporated todryness giving intermediate V (950 mg, 94% yield).

Step 5: Reduction.

A suspension of product V (5.76 grams) in Methanol (72 mL) was stirredat 0° C. and NaBH₄ (730 mg) was added. After 2 hours the reaction wascompleted, and the solvent was removed under reduced pressure. The crudeproduct was suspended in 30 mL of water and extracted with CH₂Cl₂. Theorganic layer was separated and dried over Na₂SO₄ and evaporated todryness. The crude solid was purified by flash chromatography (SiO₂,Cyclohexane/AcOEt 7/3 as eluent) giving the product VI (5.16 grams, 89%yield).

Step 6: Ketone Deprotection.

To a stirred solution of product VI (2.85 grams) in 350 mL of distilledAcetone, P-toluenesulfonic acid (7.14 grams) was added. After 3 hours atroom temperature a 5% solution of NaHCO₃ was added and the solvent wasremoved under reduced pressure. The product was extracted with CH₂Cl₂.The organic layer was dried over Na₂SO₄ and evaporated to dryness,giving the intermediate VII (2.13 grams, 95% yield).

Step 7: Synthesis of PST 3093.

To a stirred solution of 6β-hydroxyandrostane-3,17-dione VII (4.5 grams)in THF (113 mL), a solution of O-(Carboxymethyl)hydroxylaminedihydrochloride (1.56 gram) in H2O (5 mL) was rapidly added dropwise.After 1.5 h at room temperature under vigorous stirring, NaCl (6.4grams) was added and the mixture stirred for 15 min. The phases wereseparated, and the aqueous phase was extracted three times with THF (50mL). The combined organic extracts were dried over Na2SO4, filtered andevaporated to dryness giving 5.95 grams of crude product.

The crude product was purified by flash chromatography (SiO₂,CH₂Cl₂/MeOH/Acetic acid, 92.5/7/0.5) giving(E,Z)-[(6-beta-hydroxy-17-oxoandrostan-3-ylidene)amino]oxyacetic acid,PST3093 (3.7 grams, 69% yield).

EXAMPLE 2 Biological Activity of Istaroxime Metabolites ProceduresAnimal Care

The investigation adheres to the Guide of the Care and Use of LaboratoryAnimals published by the National Institute of Health (NIH publicationNo. 85-23, revised 1996) and to the guidelines for animal care endorsedby the participating institutions.

Purification of Dog Renal Na,K-ATPase and Na,K-ATPase Activity Assay

Purification of renal Na,K-ATPase was performed according to the methodof Jørgensen (Methods Enzymol. 1988; 156:29-43). Kidneys were excisedfrom 1-3 year-old male beagle dogs (WuXi AppTec, Suzhou Co., Ltd. 1318Wuzhong Ave., Wuzhong District Suzhou, 215104 P.R. China) underpenthobarbital anesthesia (Import Authorization from Italian HealthMinistry 0009171-09/04/2015-DGSAF-COD_UO-P, 2015). Kidneys were slicedand the outer medulla was dissected, pooled and suspended (1 g/10 ml) ina sucrose-histidine solution, containing 250 mM sucrose, 30 mM histidineand 5 mM EDTA, pH 7.2 and homogenized. The homogenate was centrifuged at6.000 g for 15 min, the supernatant was decanted and centrifuged at48.000 g for 30 min. The pellet was suspended in the sucrose-histidinebuffer and incubated for 20 min with a sodium-dodecyl-sulphate (SDS)solution dissolved in a gradient buffer, containing 25 mM imidazole and1 mM EDTA, pH 7.5. The sample was layered on the top of a sucrosediscontinuous gradient (10, 15 and 29.4%) and centrifuged at 60.000 gfor 115 min. The pellet was suspended in the gradient buffer.

Na,K-ATPase activity was assayed in vitro by measuring the release of³²P-ATP, as described previously (see Ferrandi M. et al., Hypertension1996; 28(6):1018-25). Increasing concentrations of the standard ouabain,or tested compound, were incubated with 0.3 μg of purified dog kidneyenzyme for 10 min at 37° C. in 120 μl final volume of a mediumcontaining 140 mM NaCl, 3 mM MgCl₂, 50 mM Hepes-Tris, 3 mM ATP at a pH7.5. Then, 10 μl of incubation solution containing 10 mM KCl and 20 nCiof ³²P-ATP (3-10 Ci/mmol, Perkin Elmer) was added, and the reaction wascontinued for 15 min at 37° C. The reaction was then stopped byacidification with 20% v/v ice-cold perchloric acid. ³²P was separatedby centrifugation with activated Charcoal (Norit A, Serva) and theradioactivity was measured. The inhibitory activity was expressed aspercent of the control samples carried out in the absence of ouabain ortested compound. The concentration of compound causing 50% inhibition ofthe Na,K-ATPase activity (IC₅₀) was calculated by using a multipleparameter non-linear regression best fitting program (Kaleidagraph™,Sinergy Software).

SERCA2a Activity Measurement in Heart Sarcoplasmic Reticulum (SR)Microsomes

Male beagle dogs were used for obtaining cardiac tissues forSERCA2a-enriched sarcoplasmic reticulum preparations. Healthy dogs wereutilized for obtaining the data in Table 3. Chronic heart failure wasinduced in dogs by multiple intracoronary microembolizations withpolystyrene latex microspheres (45-90 mm, Polysciences, Warrington, Pa.,USA) as described previously (see Sabbah H N et al., Am J Physiol. 1991;260:H1379-84). The experiments were conducted in the GeneralPharmacology Department of Sigma-Tau, Rome, Italy.

Left ventricle tissues were dissected, homogenized in 4 volumes of 10 mMNaHCO₃ (pH 7), 1 mM PMSF, 10 μg/ml Aprotinin and Leupeptin andcentrifuged at 12.000 g for 15 minutes, as described in Nediani C. etal. (J Biol Chem. 1996; 271:19066-73). Supernatants were filtered andcentrifuged at 100.000 g for 30 min. Contractile proteins were extractedby suspending the pellets with 0.6 M KCl, 30 mM Histidine, pH 7 andfurther centrifugation at 100.000 g for 30 min. Final pellets werereconstituted with 0.3 M Sucrose, 30 mM Histidine, pH 7.

SERCA2a activity was measured in vitro as ³²P-ATP hydrolysis atdifferent Ca2⁺ concentrations (100 to 3000 nM) in the absence andpresence of the tested compounds as described previously (see MichelettiR. et al., Am J Card 2007; 99:24A-32A). Increasing concentrations ofeach compound (from 0.05 to 300 nM) were pre-incubated with 2 μg ofmicrosomes for 5 min at 4° C. in 80 μl of a solution containing 100 mMKCl, 5 mM MgCl₂, 1 μM A23187, 20 mM Tris, pH 7.5. Then, 20 μl of 5 mMTris-ATP containing 50 nCi of ³²P-ATP (3-10 Ci/mmol, Perkin Elmer) wasadded. The ATP hydrolysis was continued for 15 min at 37° C. and wasstopped by acidification with 100 μl of 20% v/v ice-cold perchloricacid. ³²P was separated by centrifugation with activated charcoal (NoritA, SERVA) and the radioactivity was measured. SERCA2a-dependent activitywas identified as the portion of total hydrolytic activity inhibited by10 μM cyclopiazonic acid (see Seidler N W. et al., J Biol Chem. 1989;264:17816-23).

Dose-response curves were fitted by using a sigmoidal curve fittingsoftware and the maximal velocity (Vmax) activity and the Kd Ca2⁺ werecalculated (Synergy Software KaleidaGraph 3.6).

Drug Toxicity Studies in Mice

Acute toxicity has been determined in the mouse (Albino Swiss CD-1, bodyweight 30 g). Mice were orally treated, or intravenously injected, withsingle administration of increasing doses of the test substance toidentify the dose causing 50% mortality. Mortality occurred within 30min after the administration and survival after 24 h. The acute toxicity(LD₅₀) was then assessed.

Haemodynamics in Streptozotocin Diabetic Rat (Echocardiography2M-Doppler-Tissue Doppler)

Sprague Dawley male rats (150-175 g) were made diabetic by a singleinjection into the tail vein of a solution of streptozotocin (STZ, 50mg/kg, Sigma-Aldrich), freshly prepared in 0.1 M sodium citrate buffer,pH 4.5. Control rats received citrate buffer. Fasting glycaemia wasmeasured after 1 week and rats with values greater than 400 mg/dl wereconsidered diabetic.

Eight to nine weeks after STZ injection, rats were submitted totransthoracic echocardiographic and Doppler evaluation performed underpentobarbital anesthesia. Two-dimensionally guided M-mode recordingswere used to obtain short-axis measurements of left ventricularend-diastolic diameter (LVEDD), left ventricular end-systolic diameter(LVESD), posterior (PW) and septal (SW) diastolic wall thicknessaccording to the American Society of Echocardiography guidelines (Lang RM et al., Eur J Echocardiography 2006; 7:79-108). Fractional shorteningwas calculated as FS=(LVEDD−LVESD)/LVEDD. Relative wall thickness wascalculated as PWTd+IVSTd/LVEDD.

Mitral inflow was measured by pulsed Doppler at the tips of mitralleaflets from an apical 4-chamber view to obtain early and late fillingvelocities (E, A) and deceleration time of early filling velocity (DT).The deceleration slope was calculated as E/DT ratio. The mitraldeceleration index was calculated as DT/E ratio.

Tissue Doppler Imaging (TDI) was evaluated from the apical 4-chamberview to record septal mitral annular movements, i.e., peak myocardialsystolic (s′) and early and late diastolic velocity (e′ and a′).

The compound PST 3093 was iv administered to STZ injected rats at thedose of 0.22 mg/kg and echocardiographic parameters were measured after15 and 30 min from the beginning of iv infusion and 10 min afterinterruption of infusion.

Statistical Analysis

Data are reported as mean±SD, as indicated. Statistical analysis wasperformed by Student's t-test (pair t test for STZ rats). P<0.05 wasregarded as statistically significant.

Biological Results In Vitro Screening Inhibition of Dog RenalNa,K-ATPase Activity

Table 2 shows the inhibitory effect of the tested compounds on theenzymatic activity of the purified dog renal Na,K-ATPase. Thecorresponding IC₅₀ are expressed in μM concentration. Istaroximeinhibited the Na,K-ATPase activity with an IC₅₀ of 0.14 μM, similar tothat of Digoxin, while PST 2915 inhibited the Na,K-ATPase activity withan IC₅₀ of 2.1 μM. Further, PST 3093 did not significantly inhibitNa,K-ATPase activity at all (IC₅₀>100 μM).

TABLE 2 Inhibition of dog renal Na,K-ATPase. Compound IC₅₀, μM DIGOXIN 0.18 ISTAROXIME  0.14 3093 >100 2915 2.1 2922 >100SERCA2a ATPase Activity in Heart-Derived SR Microsomes from Normal Dog

The compounds disclosed herein were tested on SERCA2a ATPase activityprepared from normal and failing dogs in a range of concentrations from0.1 and 500 nM. The effect has been expressed as % increase of the Vmaxactivity of a control sample run in the absence of compound. Data aremean±SD, where n indicates the number of experiments.

In the normal dog SR vesicles, istaroxime, PST 3093 and PST 2915significantly stimulated SERCA2a activity at concentrations of 0.1 nMand 10 nM (see Table 3). SERCA2a activation by istaroxime, PST 3093 andPST 2915 was also tested in failing dog preparations. This effect wasparticularly evident in the failing preparations (data not shown), whereSERCA2a activity is known to be depressed compared to a normal heart(Bers D M, Physiology 2006; 21:380-387), and therefore implies that thecompounds istaroxime and PST 3093 may correct SERCA2a alteration in thefailing heart.

In contrast, previous studies showed that Digoxin failed to stimulateSERCA2a activity (Rocchetti M et al., J Pharmacol Exp Ther 2005;313:207-215; Ferrandi M et al., Br J Pharmacol 2013; 169:1849-61).

TABLE 3 SERCA2a ATPase activity in heart-derived SR microsomes fromnormal dog. Data are expressed as % increase vs control and are mean ±SD Vmax % increase (μmol/min/ vs control Concentration mg prot) *p <0.05; Compound nM (ng/ml) mean ± SD **p < 10.01 Istaroxime 0 1.252 ±0.083 (n = 5) 0 0.1 nM (0.039 ng/ml) 1.423 ± 0.123 (n = 5) 14* 10 nM(3.9 ng/ml) 1.505 ± 0.111 (n = 5) 20** 3093 0 1.312 ± 0.050 (n = 5) 00.1 nM (0.037 ng/ml) 1.641 ± 0.194 (n = 4) 25** 10 nM (3.7 ng/ml) 1.556± 0.106 (n = 4) 19** 2915 0 1.312 ± 0.050 (n = 5) 0 0.1 nM (0.041 ng/ml)1.464 ± 0.184 (n = 5) 11 10 nM (4.1 ng/ml) 1.526 ± 0.038 (n = 5) 16*2922 0 1.312 ± 0.050 (n = 5) 0 0.1 nM (0.037 ng/ml) 1.466 ± 0.112 (n =5) 12* 10 nM (3.7 ng/ml) 1.541 ± 0.170 (n = 5) 17*

In Vivo Studies Acute Toxicity in Mouse

The acute toxicity of the tested compound PST 3093 was determined in themouse (Albino Swiss CD-1, body weight 30 g). Compound PST 3093 wasorally administered or intravenously injected at increasing doses toidentify the dose causing 50% mortality. Mortality occurred within 30min after the administration and survival after 24 h.

The results for PST 3093 acute toxicity are reported in Table 4 andindicated that the compound had an LD₅₀>250 and 200 mg/kg after iv ororal administration, respectively. For comparison, the acute toxicityfor the reference compound istaroxime has been also included in Table 4.

TABLE 4 Acute toxicity (LD₅₀) of Istaroxime and PS3093 in mouse.Compound LD₅₀ mg/kg lstaroxime i.v. 29-32 lstaroxime os 200 3093i.v. >250  3093 os  >200  i.v., intravenous os, oral

Haemodynamics in Streptozotocin (STZ) Diabetic Rats (Echocardiography2M-Doppler-Tissue Doppler)

Table 5 shows the echocardiographic parameters in STZ diabetic ratsbefore and after 15 and 30 min from iv infusion of PST 3093 at 0.22mg/kg, and 10 min after interruption of infusion. Data is presented asmean±SD, and values with an asterisk are statistically significant withat least p<0.05.

The data indicated that in an animal model characterized by a diastolicdysfunction, such as the STZ diabetic rats, PST 3093 administrationameliorated diastolic function. In particular, E wave (which representsthe early filling velocity of transmitral inflow during the rapidfilling phase and constitutes the energy dependent phase of LVrelaxation, mainly mediated by SERCA2a activity) was significantlyincreased in STZ rats at 15 and 30 min after PST 3093 infusion (Table5). This effect was consistent with a stimulation of SERCA2a activity bythe compound, as shown in the in vitro assay (Table 3), suggesting theability of PST 3093 to restore SERCA2a function activity, which isdepressed in STZ rats as shown by Choi et al. (AJP 2002; H1398-H1408).

However, it should be considered that the peak E velocity is influencedby the preload and is directly correlated with heart rate (HR) (Mihm M Jet al., Life Sci. 2001; 22; 69(5):527-42; do Carmo J M et al., AJP 2008;295:H1974-1981). Conversely, the deceleration time of E wave (DT), andthe related changes in mitral deceleration index (DT/E) and decelerationslope of E wave (E/DT), are not affected by HR changes and areconsidered robust indicators of diastolic function and early signs ofdiastolic dysfunction (Mihm M J et al., Life Sci. 2001; 22;69(5):527-42). In particular, the behaviour of some echocardiographicdiastolic parameters, such as the DT of E wave, can be affected even inopposite direction according to the various grades of diastolicdysfunction. As clearly indicated in previous publications (see Mitter SS et al., JACC 2017; 69(11):1451-1464), the DT of E wave is usuallyprolonged when diastolic dysfunction is of grade 1 and becomes veryshort in patients with grade 3 diastolic dysfunction, being dynamicallyaffected also by the state of pulmonary congestion of the patient.

In this respect, the variation of the DT of E wave in animal models ofdiastolic dysfunction, as compared to the healthy controls, may vary inopposite directions. For example, in rats with diabetic cardiomyopathyinduced by STZ injection, the DT results are equal to (see Thackeray J Tet al., Cardiovasc Diabetol. 2011; 10:75; Carillion A et al., PloS One2017; e0180103) or even longer than in control rats (Joffe I I et al.,JACC Vol. 34, No. 7, 1999; 2111-2119; Guido M C et al., Oxid Med CellLongev. 2017; 5343972); while in dogs with heart failure induced bymicroembolization of the coronary arteries, the DT results are reducedas compared with control healthy dogs (Sabbah H et al., Am J Cardiol.2007; 99(2A):41A-46A). Furthermore, unlike transmitral Doppler flow,tissue-Doppler (TDI) parameters were relatively unaffected by load and adecrease in the early relaxation velocity (e′) would be an unequivocalindicator of diastolic dysfunction.

The data evidenced marked effects of PST 3093 on these parameters. The Ewave is significantly prolonged by PST 3093 treatment, while the DT isreduced. A significant reduction of DT and DT/E with increased of E/DTand e′ are shown in Table 5. The increase of e′ appeared to beassociated with a significant increase of CO and SV, while nosignificant change of heart rate was observed. Of note, the direction ofthe effects of PST 3093 on DT and E/e′ are the same as those obtainedwhen this STZ rat model was treated with Istaroxime.

In contrast, in the HF dog model of coronary microembolization, wherethe DT was reduced as compared to control dogs (Sabbah H et al., Am JCardiol. 2007; 99(2A):41A-46A), the effect of istaroxime was to prolongthe DT. In other words, independently from the variation of the DT of Ewave in the HF animal models as compared with their respective controls,istaroxime reverses such parameters toward the levels present in thecorresponding control animal and improves diastolic function.

These effects were more evident after 30 min from the beginning of PST3093 infusion and tended to disappear after 10 min from the interruptionof infusion. The effects of PST 3093 on the impaired cardiac function ofSTZ rats were consistent with the SERCA2a stimulatory activity of thecompound that, by correcting the depressed cardiac relaxation, increasedthe amount of blood available for contraction and resulted in anincrease of volume of blood pumped from the ventricle (SV).

To evaluate the relevance of the above results to the human condition,the obvious pathophysiological differences between the patients with AHFand the STZ rats should be considered. In the latter, marked changes inthe body fluids, sympathetic nervous system and heart rate, may, per se,affect echocardiographic parameters, independently from the changes incellular Ca²⁺ handling and decrease of SERCA2a activity (Mihm M J etal., Life Sci. 2001; 22; 69(5):527-42; do Carmo J M et al., AJP 2008;295:H1974-1981). Therefore, the similarities between human and rats inDT/E, E/DT and e′ changes may be considered as having the sameunderlying mechanism—a stimulation of the SERCA2a activity by PST 3093.

TABLE 5 Haemodynamic parameters after 3093 iv infusion in STZ diabeticrats STZ 3093 STZ 3093 STZ 3093 after 0.22 mg/kg iv 0.22 mg/kg iv 10 minfrom Echo STZ before after 15 min after 30 min STOP infusion FunctionParameter (n = 10) (n = 10) (n = 10) (n = 8) E 0.91 ± 0.197 1.048 ±0.218* 1.076 ± 0.211* 0.89 ± 0.19 Diastolic DT 48.5 ± 12.01 40.2 ± 9.5436.6 ± 6.09* 41.88 ± 7.86* function DT/E 56.72 ± 20.46 40.39 ± 15.04*35.47 ± 10.19* 49.08 ± 13.09* E/DT 20.61 ± 9.91 27.51 ± 8.42* 30.36 ±8.73* 22.36 ± 8.89* E/e′ 39.55 ± 5.43 38.61 ± 4.71 38.7 ± 4.26 39.45 ±4.18 e′ 22.94 ± 3.35 27.01 ± 3.4* 27.77 ± 4.27* 22.48 ± 3.35 CO 187.7 ±43.35 231.7 ± 64* 230.6 ± 46.7* 200.88 ± 51.7 overall HR 270 ± 54 271 ±33 269 ± 29 249 ± 28 SV 0.7 ± 0.15 0.85 ± 0.18* 0.86 ± 0.14* 0.81 ±0.17* *P < 0.05 compared to basal values E, E wave, early fillingvelocity of mitral inflow DT (ms), deceleration time of E wave DT/E(S²/m), mitral deceleration index E/DT (m/s²), decelaration slope E/e′,index of LV filling pressure e′ (cm/s) TDI, early relaxation velocity CO(ml/min), cardiac output HR (beat/min), heart rate SV (ml/beat), strokevolume

1. A method of treating acute heart failure in a subject in needthereof, comprising the steps of: administering to the subject a dose ofat least 0.2 mcg/kg/min of istaroxime; wherein the administeringcomprises intravenous infusion of the istaroxime for an infusion periodlonger than 6 hours; and measuring or observing one or more parametersof heart function, wherein the one or more parameters includes diastolicfunction; wherein the administering of the istaroxime by intravenousinfusion for an infusion period longer than 6 hours results in animprovement in the one or more parameters of heart function in thesubject, as compared with administration of the istaroxime byintravenous infusion for 6 hours or less, thereby treating the acuteheart failure.
 2. The method of claim 1, wherein the improvement in theone or more parameters of heart function in the subject is determined bycomparing the parameter measured or observed in the subject at the endof the infusion period with the same parameter measured or observed inthe same subject at or before 6 hours of the infusion period.
 3. Themethod of claim 1, wherein the improvement in the one or more parametersof heart function in the subject is determined by comparing theparameter measured or observed in the subject at the end of the infusionperiod with the same parameter measured or observed in a multi-subjectcohort wherein istaroxime was administered for 6 hours or less, andfinding a statistically significant difference.
 4. The method of claim1, wherein, prior to treatment, the subject exhibits one or more of:dyspnea at rest or minimal exertion; need for intravenous diuretictherapy; systolic blood pressure less than 125 mmHg, without signs orsymptoms of hypoperfusion; reduced ejection fraction (HFrEF); E/Ea ratiogreater than 10; Brain Natriuretic Peptide (BNP) concentration of 350pg/mL or greater; and N-terminal-pro-BNP of 1400 pg/mL or greater. 5.The method of claim 1, wherein the parameters of heart function areselected from one or more of: change from the beginning or 6 hours ofthe infusion period to the end of the infusion period of Echo-Dopplerparameters selected from: (i) LV Ejection fraction (EF); (ii) LV endsystolic and end diastolic volumes; (iii) Stroke volume index (SVI);(iv) E, A and E/A ratio; (v) E/Ea; (vi) Sa; (vii) Da; and (vii) Aa;changes in dyspnea, in subjects exhibiting dyspnea prior to treatment;changes in Brain Natriuretic Peptide (BNP) or N-terminal-pro-BNP;proportion of subjects with hospital readmissions or emergency visitsfor cardiovascular reasons within a pre-determined time period aftertreatment; proportion of subjects with episodes of worsening heartfailure; and length of the hospitalization.
 6. The method of claim 1,wherein the one or more parameters of heart function are measured orobserved before beginning the infusion period, or at selected timesduring the infusion period, or at the conclusion of the infusion period,or at a selected time after the infusion period.
 7. The method of claim1, wherein the infusion period is selected from: at least about 12hours; at least about 24 hours; at least about 36 hours; and at leastabout 48 hours.
 8. The method of claim 1, wherein the infusion period issufficient to permit accumulation of a plasma concentration greater thanabout 5 ng/ml of an istaroxime metabolite selected from PST 2915(Formula II), PST 3093 (Formula III), or a combination thereof.
 9. Themethod of claim 1, wherein the istaroxime is administered at a dose ofabout 0.2 mcg/kg/min to about 1.5 mcg/kg/min.
 10. The method of claim 9,wherein the istaroxime is administered at a dose of about 0.25mcg/kg/min to about 1.0 mcg/kg/min.
 11. The method of claim 10, whereinthe istaroxime is administered at a dose of about 0.5 mcg/kg/min orabout 1.0 mcg/kg/min.
 12. The method of claim 1, wherein the subjectexhibits acute heart failure with preserved ejection fraction (HFpEF) ormid-range ejection fraction (HFmEF).
 13. The method of claim 12, whereinthe istaroxime is administered at a dose of about 1.0 mcg/kg/min orless, and the infusion period is at least about 24 hours.
 14. The methodof claim 1, wherein the dose of istaroxime is changed one or more timesduring the infusion period.
 15. The method of claim 14, wherein the doseof istaroxime is reduced after about 6 hours of infusion.
 16. The methodof claim 1, wherein the treating results in improved diastolic function.17. The method of claim 16, wherein the treating results in absence ofsignals of increased arrhythmias.
 18. A method of treating acute heartfailure in a human subject, comprising administering to the subject byintravenous infusion a pharmaceutical composition comprising istaroximeat a dose of about 0.25 mcg/kg/min to about 1.0 mcg/kg/min, for aninfusion period of at least about 24 hours, wherein the administering ofthe istaroxime results in an improvement in diastolic function in thesubject, thereby treating the acute heart failure.
 19. The method ofclaim 18, wherein the treating results in absence of signals ofincreased arrhythmias.
 20. The method of claim 18, wherein the dose ofistaroxime is changed one or more times during the infusion period. 21.The method of claim 20, wherein the dose of istaroxime is reduced afterabout 6 hours of infusion.
 22. A pharmaceutical composition comprisingistaroxime for intravenous infusion in a human individual for aninfusion period of longer than 6 hours, formulated to administer a doseof istaroxime of about 0.2 mcg/kg/min to about 1.5 mcg/kg/min over theinfusion period.
 23. The pharmaceutical composition of claim 22,formulated to administer the dose of istaroxime over an infusion periodselected from: at least about 12 hours; at least about 24 hours; atleast about 36 hours; and at least about 48 hours.
 24. Thepharmaceutical composition of claim 22, formulated to administer thedose of istaroxime over an infusion period sufficient to permitaccumulation of a plasma concentration greater than about 5 ng/ml of anistaroxime metabolite selected from PST 2915 (Formula II), PST 3093(Formula III), or a combination thereof.
 25. A kit comprising: (1)istaroxime in a pharmaceutical composition formulated for intravenousinfusion; and (2) instructions for administering the pharmaceuticalcomposition to a human individual having heart failure, comprisinginfusing the istaroxime at a dose of about 0.2 mcg/kg/min to about 1.5mcg/kg/min for an infusion period longer than about 6 hours.
 26. The kitof claim 25, wherein the instructions call for an infusion periodselected from: at least about 12 hours; at least about 24 hours; atleast about 36 hours; and at least about 48 hours.
 27. The kit of claim25, wherein the istaroxime is administered at a dose of about 0.25mcg/kg/min to about 1.0 mcg/kg/min.
 28. The kit of claim 25, wherein theistaroxime is administered at a dose of about 0.5 mcg/kg/min or about1.0 mcg/kg/min.
 29. The kit of claim 25, wherein the instructions callfor changing the dose of istaroxime one or more times during theinfusion period.
 30. The kit of claim 29, wherein the instructions callfor reducing the dose of istaroxime after about 6 hours of infusion.